Science topics: ChemistryBiochemistryBiochemical MethodsAnalytical ChemistryAnalytical Chemistry TechniquesProtein PurificationHis Tag PurificationScience methodHis Tag Purification - Science methodExplore the latest questions and answers in His Tag Purification, and find His Tag Purification experts.Questions (199)Publications (17)Questions related to His Tag Purification2 Shamita Mahzabinasked a question related to His Tag PurificationWhy might I get more protein in elution 3?Question6 answersOct 22, 2015after purifying my HIS tagged protein on IMAC , when i calculated protein concentration, i get very high concentration of protein on elusion 3 (for convenience,i got highest concentration of protein in elusion 1, lowest concentration in elusion 2) . also on sds page analysis, i saw there is thick band in both elusion 1 3, and a thinner band in elusion 2. can anyone suggest, how i might get this unusually high concentration of protein in elusion 3 (which should naturally contain the least amount of protein compared to elusion 1 2). thanks in advance :) shamita.sds gel - Copy.png3.11 MBRelevant answerAneesh ChandranOct 27, 2015AnswerDear Shamitha,In first two elutes it will be the wash buffer coming out which is there in the column. Once the wash buffer fully flushed out then only the elution buffer stat coming out which have of course more proteins than the previous elutes.View0 Recommendations Hai Liasked a question related to His Tag PurificationHow can I further purify my protein?Question11 answersOct 22, 2015I am working on a membrane protein. After I mix the lysate from the solubilization of membrane from pichia yeast and nickel beads, I washed thoroughly with 20 mM and 40 mM imidazole, and elute with 350 mM imidazole. After I ran the SDS-PAGE, i saw a major band and many other upper bands. The purity is only about 50%. I want to further purify my protein.First I tried to use the Q column. But I am not able to separate the major peak and other peaks.Second, i tried to add a TEV site before His8. I cut with TEV, and further load into Nickel column. However, the cut protein is still not pure.Can anyone suggest anything to improve the purity of my protein to 80%? Can i load my his-tag protein into the second nickel column to further purity my protein? thanks.Relevant answerDominique LigerOct 23, 2015AnswerHi there,You might consider more steps in your protocol: going from 40mM to 350mM imidazole  is a big step. Either you could include more steps at 100 an 200mM (for instance) or set up a gradient of imidazole to increase the resolution.View22 Recommendations Charles Zogzasasked a question related to His Tag PurificationHas anyone experienced any metal binding properties of GST fusion protein?Question3 answersOct 19, 2015I am using a GST-fusion construct for a metal-binding protein that we work on. We avoid a traditional 6x-His tag because Histidine residues have a fairly strong affinity for metal ions (hence, why  Ni++ beads are able to pull out the tagged protein), and since we are analyzing a metal binding protein we do not want any interference from the Tag. However, our assays seem to indicate that our negative binding control (just GST alone) is binding small amounts of metal. Has anyone experienced anything like this or know of any literature that might delve into this issue a bit deeper? Thank you in advance. Relevant answerPierre BéguinOct 20, 2015AnswerRemoving the GST  tag would also solve your problem, if your construct provides a protease processing site.View0 Recommendations Bahja Al-Riyamiasked a question related to His Tag PurificationWhat can I do to improve my protein purification?Question12 answersOct 20, 2015I am trying to purify a protein for crystallization purpose, so I need an ultra-pure product. I expressed the protein as MBP-GFP fusion with His tag (at the C-terminal). The purification scheme was affinity chromatography (His column) followed by overnight digest with TEV protease (to cleave MBP-GFP-His), then cation exchange chromatography and finally size exclusion chromatography. The product I got at the end was my 47 kDa protein (most concentrated and seen as a blob in SDS gel) plus 3 other proteins (35 kDa, 30 kDa and 12 kDa) as shown by SDS-PAGE gel. I sent the bands at 35 kDa and 12 kDa for mass spectrometry analysis, and as expected it was a degradation product of my protein of interest. Basically, the N-terminal part of the protein was cleaved after TEV digest. Peptide cutter software did not show any clear cleavage sites in my protein, what can be done to avoid this degradation? Would mutating a residue at the cleavage site help solve this problem? The band at 30 kDa is most probably TEV protease (as I had the same problem with other protein before and mass spectrometry analysis showed that it is TEV protease). I do not understand how TEV protease is not separated from my protein after ion exchange and gel filtration? It shouldn t be hard to separate since it has a pI of 9, and therefore it should not bind to ion exchange column with the buffers that I used! Did anyone face this problem of residual TEV protease? How did you solve the problem?P.S. I tried to express my protein without the N-terminal domain (which was cleaved) to get a more stable product but it wasn t expressed at all. I also tried other truncations with no avail (zero expression!). It seems that this protein can only be expressed as full length.I would appreciate any help/suggestions for the above problems, as I have already spent 1 year and a half working on this protein!Thank you!Relevant answerPaul C EngelOct 20, 2015AnswerWhy so many tags - MBP and GFP and poly-His?Your 35kDA and 12kDa fragments evidently represent a single cleavage site. Does the sequence at this site in any way match the sequence specificity of the TEV protease? In theory you could of course, as you suggest, eliminate the cleavage by changing the amino acid at that position, but a) it seems a lot of extra effort; b) it might cause other problems with folding or the activity of your protein; c) maybe you could eliminate the problem more simply.  If it is the TEV protease that is the guilty party could you treat either with a smaller amount (also helping with your 30kDa band problem) or for a shorter time? If it is not the TEV protease then maybe a cocktail of inhibitors of other proteases might prevent this cleavage.  As for the contamination, maybe a different type of chromatography - hydrophobic?PaulView24 Recommendations Luiza Mendonçaasked a question related to His Tag PurificationAny advice on non-denaturing Protein concentration?Question8 answersOct 19, 2015We have a pretty expensive protein solution at 0.7ug/ml (total of 3ug), that we need to concentrate till we get to 3ug/ml. We can t waste any ug of it, and we can t denature it. The protein is His-tagged and has 24KDa. I know a Ni column seems an obvious choice, but as I said, I can t waste any ug, and in the past, I had a lot of waste using Ni columns, but maybe I just chose a poor column. Any tips? Protocols, brands, hacks, anything is appreciated! Thanks!Relevant answerPierre BéguinOct 19, 2015AnswerHello LuizaI would try a small ultrafiltration cartridge,, e.g. Vivaspin 500 and pass your prep in several batches. Keeping the volume of the cartridge as small as possible should minimize losses due to adsorption to the walls and to the ultrafiltration membrane.View8 Recommendations Venkatasubramanian Vidhyasagarasked a question related to His Tag PurificationWhy is the protein inactive after gel filtration?Question5 answersOct 16, 2015Hi,I am facing problem in my purification. I have cloned the protein in pET28a vector (His-tagged) and purified it using denaturing agent, lauryl sarcosine with lysis buffer: 25 mM Tris, 150 mM KCl, 0.1% Tween 20, 0.1% sarcosine. After loading the lysate on to Ni-NTA beads, the beads were washed with lysis buffer without sarcosine followed with 25 mM Tris, 500 mM KCl, 0.1% Tween 20 and 25 mM Imidazole. The protein was eluted with 25 mM Tris, 500 mM KCl, 0.1% Tween 20, 10% glycerol and 250 mM Imidazole. The fraction, although not so pure, exhibits its activity. When we dialyse the fraction against the buffer 25 mM Tris, 300 mM KCl, 0.1% Tween 20 and 10% glycerol, we could still find the activity. However when loaded the Ni-NTA fractions on gel filtration, Sephacryl S-300HR, and eluted the fractions using dialysis buffer the activity is lost. What could be the reason. I am struggling for long period of time. Thank you.Relevant answerPaul D JonesOct 16, 2015AnswerI agree with thew protein inhibitor suggestion.  Also go thru your procedure and estimate total recovery of activity.  If you have 100000 units of activity at the start of the procedure how many units do you have coming thru each step.  This may just be a gradual and steady loss of activity over time.View8 Recommendations Sheenam Vermaasked a question related to His Tag PurificationAny advice on a protein purification problem with His tag protein and Ni -NTA column?Question13 answersOct 3, 2015We are facing purification problem with a His-tag cloned gene. The lysis buffer is 50mM tris pH- 7 , 100 mM NaCl and 1mM PMSF. The Protein (27 kda dimer) is bound to Ni-NTA Matrix and then washed with increasing conc. of imidazole upto 100 mM  and then eluted at 200 mM imidazole. Even after proper washing with 50 mM-100mM Imidazole, the final eluted protein shows a number of bands other than our expressed protein. i m using pre packed HiTrapIMAC column linked with FPLC. Expression system is Rosetta Blue as my protein contain amber stop codon. Post NI-NTA i have done GFC also, But in that case my protein is coming in void volume with all impurities. Also, yield of my protein is very less. for clarification i m attaching gel picture, the marked region is my protein of interest.Any suggestions.Thanks in advance. 20150905_231328.jpg2.32 MBRelevant answerDeepan ShahOct 9, 2015AnswerHi Sheenam,The best ratio of protein of interest to contaminant is in lanes 44/1 and 99/2. What did you do differently in those preps? Can you repeat the same? Increasing the NaCl to 0.5M and addition of imidazole to 20-40 mM in the lysis buffer will help reduce a lot of the other contamination. As others have suggested also use a column with a smaller binding capacity.It is likely that the next most prominent band in both these (the higher mw one) is a dimer (the size standards aren t defined on the picture so it s difficult to tell). If they are dimers and other multimers then you actually have quite pure protein in those two lanes. The lower molecular weight contaminants are likely to be degradation products or premature translation termination products (common when you have rare codons in your gene). These can be removed by size exclusion at a later stage.Good luck!View7 Recommendations Kelly Hsiehasked a question related to His Tag PurificationHow can I optimise protein purification (protein with his-tag) (using complete his tag purification resin by ROCHE)?Question23 answersAug 31, 2015I m purifying a protein with his-tag (N terminal 6xHis) by using cOmplete his tag purification resin (ROCHE). I ve tried several conditions but I can t seem to find the best way to purify the protein.I know that part of the problem is that the resin I m using now elutes the protein during 25-40mM Imidazole, but I still hope to find a way to optimise the purification.This is how I m purifying the protein now:1. lyse the bacteria with buffer containing 15mM imidazole (pH8)2. incubate the lysate(160mL) with cOmplete his tag purification resin(4~6mL) for 2 hours3. load the resin+lysate into column4. wash with the same buffer5. wash with buffer containing 30mM imidazole (pH8)6. elute with buffer containing 250mM imidazole (pH8)The final result of the purification will be around 80%. (through SDS-PAGE, and the impurities bands are mainly at the bottom, around 10-17 kDa ) I tried using 25mM imidazole during the lyse process, however the protein just came right off when I tried to wash it for the first time. I also tried using 40mM imidazole during the second wash process, but the loss of the protein was a bit too much. So how can I optimise it? (without changing the resin I m using now...)And... because the impurities bands are mainly at the bottom... I was wondering... could it be that I let the bacteria induction process be too long so that some of the protein broke into little pieces and happened to have the his-tag as well?Relevant answerGeoff MargisonSep 2, 2015AnswerRe-reading your first post, it might be that you already have highly purified protein and you simply want to remove those smaller contaminants?? As they all have the same his-tag, it might be impossible to achieve that  by playing with the binding and elution conditions. However, we (and others) have used Sephacryl S-200 or  similar molecular exclusion columns for this.  Ultrafiltration might work, but the difference in size (and shape) between your protein and the contaminants is  critical (indeed, as it is for selection of MEC column!)View4 Recommendations Barney .asked a question related to His Tag PurificationHow can I tell if my protein is still bound to my resin?Question4 answersAug 14, 2015I am doing IMAC protein purification with a his-tagged protein but I have been having issues purifying my protein.First, the first few times I did it, I noticed that much of the protein was found in the flowthrough after pumping it through the column using a Gilson minipump. So I ran the flowthrough onto the column multiple times but still nothing being eluted as much.Second, I thought maybe the protein that I am loading onto the column is too concentrated. So I diluted my protein sample by half and I got no bands anywhere on my SDS-PAGE. Not even in the flowthrough, not even in my eluted sample.Third, I thought that maybe my sample is not getting eluted from my resin and is still bound. I took a sample of my resin and ran it on an SDS-PAGE but still nothing.Where is my protein? Please help.PS: I am also running westerns to probe against the his-tag to make sure it is his-tagged. Relevant answerTom BenderAug 18, 2015AnswerSometimes it can help to incubate your lysate with the beads for some time on a rolling wheel to increase binding time.View5 Recommendations Robert Adamu Sheyasked a question related to His Tag PurificationWhat is the difference between anti 6xHis antibody and anti-6x C-terminal His antibodies?Question4 answersJul 31, 2015I am working on expressing a protein that has a C-terminal His tag. I want to find out whether anti 6xHis antibody and anti-6x C-terminal His antibodies will give me different specificities.Relevant answerKe-Wei ZhaoJul 31, 2015AnswerRobert:  Both antibodies were raised against 6-His peptide and doesn t care N- or C-terminus.  But different Abs from different vendors do react differently in terms of sensitivity.  So, check out yourself.View3 Recommendations Chen Shuaiasked a question related to His Tag PurificationI can not separate my target protein from fusion protein?Question5 answersJul 23, 2015Hi, everyone. I am trying to purify a protein which is 15kD. Because the fusion protein become insoluble after link with GST and sumo tag, I link the His-MBP tag with it and the fusion protein become soluble. But after I use PreScission Protease digest the fusion protein overnight in 4℃, I found it can not be digest very well. Please check  the picture 1, the left is fusion protein and the right one is after digestion production. Then I use Ni-column and superdex75 column to separate my target protein. But I found that in the flow-through Ni-column, I found there are many un-digest fusion protein in it. And un-digest fusion protein and my target protein also in the same peak from superdex 75. How can I overcome this problem? Thank you very much! 1.jpg11.31 KBRelevant answerDominique LigerJul 23, 2015AnswerHi there,You get roughly 50% digest with your condition. As the digest profile is clean you may either extend the incubation time or increase protease concentration within the limit provided by the manufacturer. The problem comes from your NiNTA and SEC experiments. Obviously the fusion protein is not retained on NiNTA, what about free MBP? As you haven t mentioned it, I guess free MBP is retained. So His tag seems hidden in the fusion protein. In SEC experiment, digested and undigested proteins are eluted together which is bad as according to SDS/PAGE they exhibit very different MW. Have you estimated the apparent MW associated to the peak (is it consistent with fusion protein MW or bigger?)? You may be confronted with oligomerization of your target protein (do you know about its behavior in solution?) or worse to aggregation...View4 Recommendations Jasmina Markulicasked a question related to His Tag PurificationPurification of His-tagged protein expressed in inclusion bodies?Question8 answersJul 22, 2015I am expressing a protein of pI 7; I have been able to optimise expression however when it comes to purification, especially at the refolding stage, I am losing a lot of protein. I understand at this stage a lot of protein can be lost, but I m trying to find a way to lose the least amount of protein (as everyone is of course!). I purify under denaturing conditions, then refold by dilution in the presence of arginine. I then concentrate and dialyse out of arginine so that I am able to purify on Ni-NTA resin. Followed by TEV cleavage then SEC for further purification. However I am also getting precipitation at the TEV cleavage stage. Does anyone have any similar problems? Should I be cleaving the His-tag before refold? Relevant answerKonrad HerzogJul 22, 2015AnswerMy personal experience with refolding is that each kind of unnatural tag, especially the his-tag is making refolding tasks even more complicated. Sometimes it depends on the location (N- or C-terminal) but generally I would try to work without his-tags in refolding procedures and I would express the target protein in its native form without any tag. You might ask why? There are many reasons, I will pick only the most important ones.- Your protein is expressed into inclusion bodies. Isolation of these inclusion bodies is a very efficient initial purification step, which is often as powerful as his-tag based IMAC, so you don t gain additional purification advantages with a his-tag.- The his-tag is a nonphysiologic charge accumulation at one side of your protein, which can have tremendously negative effects on the natural folding behavior of your protein.- The strong charge of a his-tag can attract other proteins to become part of the inclusion bodies, thus weakening the purifying effect of inclusion body isolation.- You are cleaving the his-tag, so you introduce an additional experimental step, you contaminate your liquid chromatography columns and pumps with a protease (some of your colleagues might not be that happy about this) and, as you are cleaving the tag, there seems to be no special need for it. Why are you still using it?I would recommend the following:Express your target protein without any tag. Then isolate your inclusion bodies and perform a screening in small volumes for both, optimal resolubilizing conditions and optimal refolding conditions. Follow Mathews hints regarding pH, temperature and ionic strength screening and add some additional compounds to your refolding and resolubilization solutions.What is the native localization of your protein? The background of this question is difference of cytosolic, extracellular etc. conditions. E.g. for the reconstitution of disulphide bonds it is often required to add reducing agents such as DTT, TCEP or beta-mercaptoethanol. A defined redox environment can be set up using appropriate amounts of reduced and oxidized glutathione to facilitate the formation of the correct disulphide bonds.Adding appropriate amounts of urea can often be helpful, as it supports the formation of correct secondary structures.Sometimes it is also important to add some detergent like tween or triton-x to resolubilize the hydrophobic parts of your inclusion bodies.Slow changes of chemical conditions can be achieved via dialysis and can help your protein to walk along the energetic road of folding enthalpies to the correctly folded energy minimum. This can be supported sometimes by thermal gradients from higher to lower temperatures. Keep in mind that your protein is optimized for a certain temperature, so it might be possible that a human protein from a 37C body temperature cannot be refolded efficiently at 4C.Directly after refolding I would perform ion exchange chromatography, e.g. on a Q-sepharose, as misfolded proteins often have many hydrophobic residues on their surface, so that only the correctly folded molecules will interact with the stationary phase. If you are working with large volumes, you can also directly put the sepharose beads into your refolding solution and after some mixing directly pack the column with the preloaded material.Hope that helps....KonradView25 Recommendations Liyana Azmiasked a question related to His Tag PurificationDoes anyone have a map of the p77 plasmid, possibly one with a his-tag?Question2 answersJun 18, 2015i ve inserted adhe gene in the p77 plasmid and transformed it into BL21 for expression. i wanted to know the size of the plasmid so that i can validate my size of adhe insertion. also, is there a p77 plasmid with his-tag?Relevant answerKonrad HerzogJul 21, 2015AnswerSeems as if the link has not been attached. Here is the thesis cited above:http://geb.uni-giessen.de/geb/volltexte/2003/1034/pdf/LandgrafFrank-2003-04-27.pdfView0 Recommendations Jerry Wangasked a question related to His Tag PurificationWhy is my protein both in the flow-through and unable to be eluted?Question6 answersJul 19, 2015During ni-nta purification of my his-tagged protein, I collected sample from each step and ran them on a sds-page. I see my protein band in the flow-through (thickness indicate about 30% of total protein content), as well as in the first wash. This band then becomes progressively lighter with subsequent wash. When I got to the elution step, I did not get any protein. Finally, I added sample buffer directly to the beads and ran run on the gel as well and saw my protein band (around 30% of total protein content). Here are more details:I used 100 microliter suspension of newly-charged nickel beads (50%) for a 5ml culture (= 1ml lysate).All buffers were freshly prepared:Binding buffer:20 mM Tris-HCl pH 8.00.5 M NaCl20 mM imidazoleWashing buffer: everything remains the same except 40mM imidazoleElution buffer: everything remains the same except 250mM imidazoleI incubated the protein supernatant with the beads for 1.5hr before centrifuging at 3500 rpm for 3mins and aspirating the supernatantI also incubated the beads with the elution buffer for 10 minutes before each elution step.Here is the gel image: I apologize for the poor quality. The 1st column on the left is the flow through and my band is the one at the red marker. You can see the band in the next lane, which is the first wash, followed by 2nd wash, 1st elution, 2nd elution, and the uneluted sample. The next lane with lots of protein is the flowthrough of a second protein which is very similar to the first one, you can see that once again most protein is lost in the first wash, but some still remains on the beads after elution.Thank you for your help, this problem has been bugging me all day. 20150716003.jpg207.32 KBRelevant answerAdam B ShapiroJul 19, 2015AnswerYour protein may be in an aggregated state. The aggregated protein did not bind to the column, but some of it got stuck on the column and was released by sample buffer. Go back to the expression conditions and look for conditions that produce more un-aggregated protein, such as lower expression temperature.View12 Recommendations Nisha Peterasked a question related to His Tag PurificationIs it normal that Flag tagged protein migrates slower than the same protein tagged with HIS-Flag tag?Question6 answersJun 1, 2015When my protein was tagged only with the Flag tag , it shows a shift on the western blot. However when I tag it with 6X His and Flag it comes back to the original size. Is this normal?The Flag tag was used for immunoprecipitation experiments and the HIS tag was added for protein purification from bacterial cells.Relevant answerSneha SinghJul 18, 2015Answerif u have a claevable site between your 6X his tag protein, after purification you can cleave and check the migration of band with or without his tag.plus the exp of charges, which makes flag+His-protein run better than Flag-protein is good and i second that tooView0 Recommendations Mohammad Kawsar Manikasked a question related to His Tag PurificationIs there any fixed time for removal of the His-tag?Question3 answersJul 8, 2015I am working on a protein having six histadine tag linked by a thrombin cleavage site and I keep the protein with thrombin for 1 hour at room temperature. However, I have seen some people using more time and volume of thrombin for cleavage. Is there standard protocol for this?Relevant answerPierre BéguinJul 9, 2015AnswerI concur with Dhiraj s answer. Although I prefer to use Tev protease, the principle is the same: for each protein, the strength of the proteolytic treatment has to be adapted in order to get the best compromise between  efficient removal of the tag and using needlessly large quantities of (costly) processing enzyme, not to speak of the risk of non-specific cleavage. This is easily done by  setting up small assays (20 µl containing 2-5 µg of the test protein) with various amounts of protease/and or incubation times. Removal of the tag will shift the size of the polypeptide by 2-3 kDa, which can easily be detected by SDS-PAGE (add a non-treated control for comparison). Once you find the appropriate conditons, just scale-up for preparative cleavage.View2 Recommendations Naghmeh Azadfarasked a question related to His Tag PurificationWhy dose not a His-tag fused protein eluate during eluation step in FPLC even with 2 M Imidazole concentration?Question13 answersJul 3, 2015I am purifying a His-tag fused protein by Äkta instrument from GE Healthcare. The column in 1 ml HisTrap column. after loading the lysis to the column and washing by washing buffer, i elute the protein using 500 mM imidazol concentration. After finishing the elution step, protein is still in the column and i have to increase imidazol concentration of the elution buffer up to 5 M imidazol to elute the proteins from the column . Do you know why? Also, i ordered new column but did not help. Do you have any idea?Relevant answerRajkumar DhanarajuJul 7, 2015AnswerHi there,1.It could be your protein is not at all expressed or its in insoluble form which hinder the His-tag accessibility to Nickel-NTA bead.2. It could also be the case that the protein expression is hindered by codon bias, promoter mutation, stop codon in the gene by random mutation, low expression.,etc.3.You can also look for any other components in your buffer that could chemically hinder the His-tag protein binding to Ni-NTA if your protein indeed expressed. 4.I would suggest you to do a mini induction in 5 ml or 10 ml culture and subject to SDS-PAGE, comassie staining and also silver staining (low expression) to check indeed protein is expressed are not.5. For low expression you can scale up the volume of culture or play with IPTG concentration for ideal induction and also try induction at low temperature.I hope this was useful to you to address you problem.RajkumarView0 Recommendations Abdul Mohin Sajibasked a question related to His Tag PurificationCan anybody suggest a cheap 6his tagged protein that i can use for western blot?Question3 answersJun 15, 2015I am doing western blot and looking to see band for my his tagged protein...Relevant answerAbdul Mohin SajibJul 6, 2015AnswerThanks a lotView0 Recommendations Hana Muheisenasked a question related to His Tag PurificationHow to remove His tag in the cloning result, can I use pACYCDuet-1 vector?Question8 answersJun 25, 2015I will use Nco1 Not1 restriction enzymespACYCDuet-1.pdf179.21 KBRelevant answerHana MuheisenJun 25, 2015AnswerHi Arthur,Thank you very much, i will clone another gene into the plasmidCheersHana aView0 Recommendations Jeremy Garzaasked a question related to His Tag PurificationHow can I detect his-tagged proteins in polyacrylamide gels?Question6 answersJun 5, 2015I m looking for info on how to stain histidine tags in SDS-PAGE gels directly. I found kits from Life Tech and Pierce but if something similar could be prepped in house that would be nice (not sure how they work either,perhaps anti-his w/ fluorescent probe?) .Where could I find a technique/protocol for probing polyacrylamide gels if I purchased a fluorescently tagged anti-his myself? Also what considerations would there be, it s polyhis but would this work only for native gels?Relevant answerAdam B ShapiroJun 6, 2015AnswerYou could use the gel to do a Western blot with anti-poly-His antibody.View27 Recommendations Nicole Innissasked a question related to His Tag PurificationDoes anyone have experience using infrared secondary antibodies for western? What protocols work best for you?Question7 answersMay 28, 2015I am trying to detect a purified bacterial his-tagged protein on western blot (using nitrocellulose membrane). I did see the protein in elution fractions on SDS gel using coomassie stain. Primary used to detect is a rabbit anti-his, and my secondary is anti rabbit-680 conjugate. I blocked with 5% milk, incubated with primary o/n at 4 in 5% milk, and also placed the secondary in 5% milk for 45min at RT (I was told by some protein loving friends that milk can also help improve secondary antibody background). I have not used these fluorescent antibodies before. Called the company and am waiting to hear back, however in short, it might be that I should tailor my staining protocol to something closer to an IF, so use serums for blocking and incubations instead of milk. Also an option to just use TBST for antibody incubations. Any help/thoughts is much appreciated! Relevant answerMartin BeckerMay 29, 2015AnswerHi Nicole,we are using an Odyssey system for performing WBs with fluorescently labelled antibodies. Here, LI-COR provides a specific blocking reagent which we use routinely and obtain good results with. I also use this reagent to dilute the antibodies. See link belowhttp://www.licor.com/bio/products/reagents/blocking_buffers/odyssey_blocker_pbs.htmlHope this might help you.View9 Recommendations Chris Brigolinasked a question related to His Tag PurificationIs it common for GST tagged proteins to be purified in a column designed for His tags?Question5 answersMay 19, 2015I am interested if two proteins have a physical interaction. One protein has a GST tag, one protein has a His tag. I put the His tagged protein into the His purification column, let it incubate, added the GST tagged protein into the column, let it incubate, and they both eluted together (lane 3). So of course I need my negative control. I use a completely new column, add only the GST tagged protein (which has already been purified in a GST column, the pre-run, purified GST tagged protein can be seen in lane 4), and then spin, do 3 washes, and two elutions. The flow through can be seen in lane 7, the first Wash can be seen in lane 8, and elutions 1 and 2 can be seen in lane 5 and 6...Obviously the GST tagged protein alone was eluted from the column with a similar affinity as when the other protein was present. Is this common? I plan on reversing it and adding the His tagged protein to the GST column next, but what are my other options? GST only has 6 total histidines (spread through its sequence) and my target protein only has 4 (spread through its sequence also).Thank you for any input! IMG_20150519_164243.jpg4.74 MBRelevant answerBryen A JordanMay 22, 2015AnswerChris, you are seeing standard and common background interactions.  The purification columns are typically made out of sticky agarose/sepharose materials which, truth be told, bind to everything to varying degrees.  You want to make sure you have conditions where you can separate out signal from noise (there will always be noise) and there are many ways to do this: Try preblocking your column with BSA, or use less protein, or use less beads (column) or increase the amount of NaCl in your buffers.  In my experience a combination of all of these works best- Just make sure you don t put too much NaCl that you inhibit the interaction between your two proteins.   View5 Recommendations Lionel Faureasked a question related to His Tag PurificationHow can I get rid of contaminants in my protein purifications?Question12 answersApr 30, 2015Hello everyone,I read a lot about protein purification on this website and I hope I could find some help with my protein purification.Background: the protein of interest is 110KDa, pI estimated at 8.2, tagged with HIS expressed in E. coli.I used different resins: NiNTA, Talon, and magnetic beads, under native or denaturing conditions. I used high concentration of salt 0.5M of NaCl, 1% of tween-20, 20mM beta-mercapto, 5mM imidazole, 1mM PMF, cocktail anti protease, and 5% glycerol, pH 7.6 in my lysis buffer. I washed very well my column with a lot of buffer (with up to 20mM of imidazole). When I elute, whatever the resin used (native or denaturing conditions) I have protein contaminants (from 90to 20KDa) in my elution fraction. I checked by mass and those proteins are not degradation of the protein of interest but various bacteria protein from E. coli. I did SEC (size exclusion chromatography), or centricon (50 or 100 MWCO) centrifugation, even second purification step using another tag added after the His tag, on the eluted fraction. I still have the contaminants. I concluded those protein contaminants are bound to the protein of interest. But how comes those contaminants are still present even when 7M of urea (or 7M of guanidine HCl) are used with the denaturing conditions?I don t know how to get rid of those contaminants. If anyone has an idea or suggestions he or she is the most welcome.Thank you very much,Regards,Lionel.Relevant answerKannan BalakrishnanMay 1, 2015AnswerTo avoid the non specific protein binds with the resin increase the salt concentration in wash buffer.Reduce the beta-mercapto concentration it will disturb the resin and protein interaction.Use the Buffer pH slightly less than this because it should be less(1or 2 ) or more compare with your PI of the protein .If you use the Ni- NTA beads in elution buffer  you can add immidazole concentration (100 to 250 mM).View10 Recommendations Charandeep Singhasked a question related to His Tag PurificationCan I purify a protein with N-term ORF-histx6-HA-ProteinA(ZZ)-Cterm on an hist-trap(Ni affinity column)? Is it only possible if hist tag is terminal?Question6 answersApr 27, 2015If during cloning my construct is N-term ORF-histx6-HA-ProteinA(ZZ)-Cterm, can I still purify the protein on hist-trap column. Will the 6-hist be still accessible to attach hist-trap column. NOTE His tag is not terminal. Thanks CharanRelevant answerDominique LigerApr 28, 2015AnswerHi there,The limitation of affinity purification is the same whatever the tag and its location in the protein sequence :will the tag be accessible to ensure specific and reversible interaction with the purification matrix?The answer to this question can t be anticipated unless you have in hand the structure of the protein.View18 Recommendations Patrick Guestasked a question related to His Tag PurificationHow can I check his-tag has been cleaved off protein?Question7 answersApr 21, 2015Hello all,I am looking into doing some recombinant protein expression experiments and due to cost would like to purify my own Tev-Protease. One thing I am not sure about is how would I check my purified protease is active? once my tagged protein is purified and I have incubated with my previously purified TEV can I visualize both the tag and the protein on the same gel (i.e./ one larger mw band prior to cleavage then two smaller bands for example?).Relevant answerAntonio ArizaApr 21, 2015AnswerAll you need is a second purification step. During the first purification step your protein will bind to the Ni-NTA via its His-tag and you elute it with an imidazole gradient. You should then dialyse your protein to remove the imidazole (this is necessary for the following steps) and then add your protease to your protein. Incubate your protein/protease mixture as required and then do the second purification step. The second step consists of passing your protein/protease mixture over Ni-NTA for a second time (this is why you need to dialyse it first, as the imidazole would interfere during this step). This time only the uncleaved protein molecules and the cleaved His-tags will bind to the column, while your cleaved protein without His-tags will go straight through it. Elute the uncleaved protein and the cleaved His-tags with imidazole.This works particularly well if your protease also has a His-tag as it will also stick to the column and therefore only pure, cleaved protein will pass through it. This is also a good protocol to follow for those proteases that rarely give you 100% cleaveage as it separates the cleaved protein molecules from those that weren t cleaved by the protease.Remember to add a small amount of imidazole (~20 mM) to your protein/protease sample before loading it onto the Ni-NTA column/beads and use a small gradient to around 50 mM imidazole to make sure your cleaved protein comes off the column/beads while the uncleaved protein and His-tags remain bound (the cleaved protein often sticks non-specifically to the column/beads without this little bit of imidazole). You can then remove the uncleaved protein and the cleaved His-tags without a gradient by passing 300 to 500 mM imidazole over the column/beads.View36 Recommendations Gina Portilloasked a question related to His Tag PurificationWhy can I not detect Flag-tagged protein anymore?Question6 answersApr 7, 2015Hi everyone!  I have transfected both HEK 293T and HEK 293 cells with my plasmid containing my gene of interest (obi-1) with the flag tag (pcDNA3.1).  When I conducted several western blots I saw my protein band (~61kDa). Now I am trying to replicate the result but it somehow stopped working. I ve tried freshly made buffers, new plasmid DNA for transfection mixes, and fresh 4X SDS-DTT loading dye (w/ bromophenol blue).To get my protein off the M2 slurry (beads: anti-flag) I have heated my dye to 72C and 100C before adding it to my protein samples, vortexed, and centrifuged before loading on SDS-PAGE.I have also preheated the dye and then incubated my protein for an additional minute, briefly vortexed, and centrifuged before loading on SDS-PAGE.Any ideas on what I can troubleshoot next?I also get a ~20kDa band detected from my transfected cells and non transfected cells. Do HEK cells make any endogenous flag-like proteins?Relevant answerElitsa Y DimovaApr 9, 2015Answerhi Gina,you plasmid might be detectable in the cells but just one point mutation within it can result in an unexpected stop codon and thus in non-fully (non-functional) expressed protein. Is your FLAG-tag N- or C-terminal? I think it will be faster and less frustrating if you sequence your plasmid before troubleshooting the western blot.Good luck!View0 Recommendations Manjula Ramuasked a question related to His Tag PurificationHow can I increase the binding affinity of GST-tagged protein to GST sepharose column?Question7 answersMar 2, 2015Hi,I am purifying a protein having GST tag. I am using 20mM tris pH 7.5, 200mM NaCl, 5mMDTT and 5% glycerol as the equlibration buffer. Column is of 2ml bed volume (I have hardly 3mg of my protein). Protein is not completely binding to the column and nearly 30% I can find in the flow through. Please suggest me how to increase the binding capacity of the protein.Thanks in advance..Relevant answerRaghothama ChaerkadyMar 2, 2015AnswerReplace 5mM DTT (competes with GSH) with 1 mM BME, include 1 mM EDTA (removes metal inhibitor of GST). During sonication do not include detergent. After sonication add 1 mM Triton X 100, mix by inversion 6 times and take supernatant for affinity purification. View63 Recommendations Ozkan Fidanasked a question related to His Tag PurificationWhat is the highest protein MW limitation for Hi-tag purification?Question5 answersFeb 11, 2015I would like to purify my protein using His-tag affinity. Does the size of protein matter in his-tag purification? What is the maximum MW of a protein that can be purified using his-tag and nickel column or beads?ThanksRelevant answerDaniel M CohenFeb 12, 2015AnswerAgreed. Size is not a factor, per se, for affinity purification. I ve used his-tags to purify protein ranging from 120 to 220kDa successfully. The challenge with larger proteins is that often they don t express well in recombinant systems. But there is no intrinsic problem with large proteins interacting with IMAC resins, provided your his tag is solvent accessible.View6 Recommendations Agnes Machadoasked a question related to His Tag PurificationCan you share your experience on enterokinase to cleave a his-tag fused protein in 2 M urea?Question3 answersJan 30, 2015Hello,My expressed protein is at 2 M urea (it will precipitate if I take urea out) and I need to cleave its his tag with bovine enterokinase.I read some references that enterokinase might be active in the presence of between 1-4 M urea, has someone had experience (enterokinase removes his-tag in 2 M urea)?Additionally, I read that for cleaving the target protein it is advisable to maintain its concentration at 1mg / mL. I could take out urea from the above mentioned sample, but then the target protein concentration could not be above 0.2 mg / mL. In this case, how do you change the contact time with enterokinase?AgnesRelevant answerOleg LaptenkoFeb 3, 2015AnswerDigest the protein when it is still on the Ni-resin under non-denaturating conditions, collect the released material and dialyze against 2M urea to re-solubilize the protein, concentrate it if needed. View18 Recommendations Natasha Kruglyakasked a question related to His Tag PurificationIs it better to express yeast kinases in E. coli or yeast to use in kinase assays?Question7 answersJan 27, 2015Hello,I have high copy yeast vectors into which are cloned yeast kinases with a N-terminal GST-His tag under the regulation of a GAL promoter. I need to purify these kinases for kinase assays, and am wondering if I should express them from the yeast vectors directly or is it better to subclone them for expression in E. coli?Thanks!Relevant answerDidier FesquetJan 29, 2015Answeras you already have  the yeast construct , and if you are famiuliar with gal induction with yeast . if you just need analytical quantities, i will first use the yeast expression system.  it is likely the kinase will be active.if you need large quantity, go the ecoli expression  system.  co express with your kinase the lambda phosphatase , this dephosphorylate the kinase and improve its expression and correct folding (see knapp lab and otherInsights into the Conformational Variability andRegulation of Human Nek2 KinaseIsaac Westwood1,2,J. Mol. Biol. (2009) 386, 476–485View16 Recommendations Raja shekar Varma Kadumuriasked a question related to His Tag PurificationSuitable expression system for 30-40 residue peptide His tag or GST ?Question4 answersDec 20, 2014Hi,I would like to express and purify a peptide of 30-40 residue long. I would like to know if I can use pET-31b vector with HIS tag or should I go with pGS-21a with GST tag. It would be helpful if any other expression systems are suggested.Thank youRelevant answerR. SelvamDec 20, 2014AnswerDear Raja Sekhar  ,I Feel pGS-21a with GST tag was better option.,Before you doing this, make sure about your peptide common purity by using RP-HPLC-LC-MS,And you first find the property of your peptide based on that only we can able to say perfectly which system is suitable.,You check the nature also, Hydrophobic peptide means the tag should need to changeFor answering this question you need to give details about your peptide, otherwise some common suggestions only possible.,Good Luck RegardsR.Selva....View18 Recommendations Roman Bonetasked a question related to His Tag PurificationHow can I remove a cleaved His-tag unspecifically bound to my protein?Question10 answersNov 13, 2014I am purifying and intrinsically disordered protein (60 residues, pI around 12) with a His tag and a 3C site. After Ni colum I cleave the tag and works fine - then I do dialysis (membrane 3500, His tag 2000 Da) and RPC C18 column (ACN gradient with 0.05% TFA), but finally I see that the His tag is still there.I ve tried to reload the sample to Ni column (in presence of 1M NaCl) but my protein only appears in the elution together with the His tag, so clearly they are sort of interacting.Any ideas in how to get rid of the His tag?Thank you very much!RomanRelevant answerRoman BonetDec 11, 2014AnswerHi people!,Just for the record - I could solve the problem and get rid off the His tag: I used an heparin column (my protein binds heparin) and did a NaCl gradient (0 to 2M) at pH=9 with 0.1% Tween20. I believe it would have worked the same using a cation exchange column. In any case, no sign of the His tag in MALDI spectra :-)Thanks for all your input again,Best wishes,RomanView5 Recommendations Jeppe Achton Nielsenasked a question related to His Tag PurificationDo you have suggestions for a crystallization exercise for high school students that includes protein expression and purification?Question9 answersDec 9, 2014Hello all,Im trying to put together an exercise for High-school level students. In this exercise I want them to express, purify and crystallize a protein.The protein should ideally be expressed with a histag/gsttag in e.coli. The protein should be able to be purified for crystallization in max. 2 steps (for example nickel column and size exclusion). The protein should also crystallize overnight.Does anyone have any good candidates? I have looked at both lysozyme and proteinase K, but I am uncertain whether or not these can be used sucessfully?With kind regards,Jeppe NielsenRelevant answerJose A. GaviraDec 9, 2014AnswerDear Jeppe, probably GST itself could be a good candidate.View10 Recommendations Zahran Somayaasked a question related to His Tag PurificationWhy can t I get a pure protein with Ni column chromatography protein purification?Question7 answersNov 28, 2014I have a problem with my protein purification, I m using the Ni column chromatography, running my cTnI protein after cleavage from the His tag to get the protein without the His tag, but it s leaking, I can t get a pure protein. Can anybody help? ThanksRelevant answerZahran SomayaDec 4, 2014Answerso it s cardiac troponin I, cleaved with Ni from the fusion partner, I added EDTA to get rid of the Ni, then dialysed it to get rid of the EDTA, and dissolved the pellet in 6M GuHCl.I got the fusion partner leaking in the flow through, so obviously, the His tag that s attached to the fusion partner isn t binding to the column.Thanks for helping.View0 Recommendations Tatyana Sysoevaasked a question related to His Tag PurificationIs it possible that a protein missing His-tag binds to Ni-NTA resin?Question9 answersNov 26, 2014I work with a protein that appears to weakly bind to Ni-resin. It binds with some efficiency under 10 mM Imidazole and starts eluting at about 50-60 mM.Relevant answerDominique LigerNov 27, 2014AnswerHi there,Of course! What is defining the affinity of proteins for NiNTA is their potential enrichment of His residues at the surface (Arg and Gln also contribute to binding). Two His residues close to each other at the surface of protein is sufficient to interact with sequestrated Nickel ion.  The function of 6xHis tag is to ensure the enrichment, the proxymity between imidazol rings and the increase in affinity as this tag is potentially able to interact with three Nickel ion. Now considering untagged protein, the natural affinity for NiNTA will be dependent on the repartition of His residues at its surface (and Arg, Gln too).View10 Recommendations Priyankar Senasked a question related to His Tag PurificationIs anyone familiar with polyproline tags for purification?Question1 answerNov 20, 2014Hello friends,I have a protein (without his-tag) naturally having one pentaproline sequence in the solvent accessible position. Does anybody have experience with purification using the help of polyproline tags?Relevant answerRavi Kant UpadhyayNov 20, 2014AnswerAtypical polyproline recognition by the CMS N-terminal Src homology 3 domain epitope tag is used that is associated interaction partner identified by Western blot. Cells are fixed and processed for immunohistochemistry using M5 and 2166.The slides are processed by an observer blind to the treatment conditions. One thousand Flag-positive on each microslip are  examined for the expression of diffuse htt and htt bodies usinga fluorescence microscope with 60 objective and an ocular grid (Eclipse TE 300; Nikon, Tokyo, Japan). The data are  statistically analyzed with Student’s t test or ANOVA and presented in bar figures as the number of cells with htt bodies per 1000 cells expressing exogenous htt. You can attach polyproline tag for recognitionpurpose on CMS N-terminalView5 Recommendations Priyankar Senasked a question related to His Tag PurificationWhy do I observe multiple peaks in flow-through?Question10 answersNov 18, 2014I am trying to purify one protein in 2mL agarose-NTA-Ni2+ column at flow rate less than 1mL/min (equilibrated in 100mM phosphate buffer pH 7.4 with 1M NaCl).Multiple peaks can be observed in flow-through. SDS-PAGE indicates first peaks contains higher and later peaks contain lesser MW proteins respectively. Any explanation?Relevant answerRicardo AzpirozNov 19, 2014AnswerRight. I m aware that it s an affinity column, but that does not eliminate the potential for sizing behavior in the beads. Either way, it s not a problem, apparently; just a curious phenomenon.View10 Recommendations Jeevanathan Kalyanasundramasked a question related to His Tag PurificationDoes Zn2+ ion need to be displaced first, before purifying zinc binding protein using IMAC chelated with Zn2+?Question4 answersNov 19, 2014Hi everyone,I am in hardship of purifying my recombinant melanosomal protein expressed in CHO cells. This protein is his tagged on the C-terminal. However, purification by Nickel and Cobalt IMAC were low for the former and slightly higher for the latter. I have recently found out that two Zn2+ ions attached to my protein s active site. I would like to exploit this for IMAC purification chelated with Zn2++. Co2++ has been shown to displace Zn2++ slightly; as well as increasing concentration of Zn also displaces bound Zn2+ ions in other Zinc binding proteins.The question is; Should I remove the occupying Zn2+ ions before proceeding to IMAC or does the increased concentration of Zn2+ in the column can be expected to attract Zinc bound protein?Thanks in advance.Relevant answerDominique LigerNov 19, 2014AnswerHi there,It has happened to me to purify Zinc binding protein on IMAC without any problem. So I think your problem of low yield might be because of hindered accessibility to the HisTag. I would suggest to insert the tag on the opposite end of the protein and have a try. If you want to deplete Zinc, you have to be careful as the Zinc might contribute to stability of the native form of your proteins and therefore depletion could promote denaturation.Hope it helps.View12 Recommendations Matthew Whitleyasked a question related to His Tag PurificationAre there useful purification tags for denaturing purification conditions?Question3 answersNov 17, 2014Hello all,I am working with a heterodimeric protein complex that I must form by expressing and purifying each component under denaturing conditions and then co-refolding the two denatured components.  Upon co-refolding, the heterodimer is soluble, whereas the two individual components are completely insoluble.  Currently, each construct has an N-terminal 6x-His tag, which I use to purify the denatured protein from the rest of the cellular contents.  The problem is that, after co-refolding, both components of the heterodimer are his-tagged, and I cannot separate intact heterodimer from residual free monomers.  This would not be a problem if I could put a different tag on one of the complex components, in which case I could simply do a sequential two column purification to separate complex from free monomers.  To do this, however, I need a tag that can be used under denaturing conditions.Does anyone have a good suggestion for a tag other than hexahistidine that can be used under denaturing conditions? Relevant answerAntonio ArizaNov 18, 2014AnswerHi Matthew,If the size of your heterodimer is sufficiently different from that of the two individual monomers, then the simplest method would be to use SEC (size exclusion chromatography, also known as gel filtration) rather than adding a second tag to one of the monomeric species. Run your refolded sample over a Superdex 75 or 200 (depending on the size of your heterodimer) and all the components will be separated according to size. Your large (molecular size, not overall volume) heterodimer pool should elute first as a separate peak from the two smaller monomer pools. This will work better if your two monomers are of a similar size, as the heterodimer will be nearly twice the size of the individual monomers. If one of the monomers is significantly smaller than the other, then the size difference between the heterodimer and the larger of the two monomers might not be enough to separate them properly.View4 Recommendations Kedar Sharmaasked a question related to His Tag PurificationHow to remove host cell protein at the time of protein recombinant protein purification?Question5 answersNov 3, 2014at the time of protein purification using Ni-NTA method almost equal amount of host cell protein is coming with recombinant protein. my protein is mesophilic in nature. please suggest me to how to remove host cell protein. Relevant answerAntonio ArizaNov 3, 2014AnswerHi Kedar,Here are my tips for a successful Ni-NTA run.Use protease inhibitors in your lysis buffer (they need to be EDTA-free as EDTA would chelate the nickel from your beads/resin/column). Add the inhibitors to the cells with the buffer before you lyse them, not after.Use at least 50 to 100 mM buffer (TRIS, or HEPES, or phosphate, etc...) at a minumum pH of 7.6 and a maximum pH of 9. Check the pI of your protein including its his-tag to make sure the pH of your cell lysate is adequate. Using less that 50 mM buffer might not actually buffer your lysate, which can often be more acidic than you realise and at a pH below 7.6 the binding of his-tagged proteins for Ni-NTA is reduced.Also use 500 mM NaCl in your buffers as most proteins are more soluble in high salt and it reduces the non-specific binding of contaminating proteins too.Add 5-10 mM betamercaptoethanol (BME) to stop your protein aggregating and other proteins from binding to your protein via disulphide bridges. You must check how much BME your nickel beads/ column can withstand as too much will reduce the nickel and result in a brown mess that will ruin your purification. Don t use DTT instead BMA as it is a stronger reducing agent and will lead to a brown mess even quicker than BME.Your lysis/binding buffers should have 10 to 30 mM imidazole as this will also reduce non-specific binding of non-his-tagged proteins to the column.As mentioned earlier, wash your column/beads stepwise with buffers containing 50, 70 and 90 mM imidazole for 6His-tagged and 50, 100 and 150 mM imidazole for 10His-tagged proteins to remove as many contaminants as possible. Finally, elute it with buffer containing either 300 (for 6His-tagged) or 500 mM imidazole (for 10His-tagged proteins). Keep all the flowthroughs and test them to see where your protein elutes, as it might not necessarily elute at 300-500 mM imidazole. Often his-tagged protein elute as early as 50 mM imidazole (i.e. when the his-tag is partially occluded), though well-behaved 6His-tagged proteins should elute around 160 mM imidazole and 10His-tagged proteins around 300 mM imidazole.You can also add 5-10% glycerol to your buffers, which helps with protein solubility.As Dominique mentioned, ion exchange and size exclusion chromatography can be used to further purify your protein, as Ni-NTA rarely gives very pure protein samples when used as a single purification step.View6 Recommendations Amit Kumarasked a question related to His Tag PurificationWhy purified protein band shifted to lower molecular weight in SDS-PAGE?Question20 answersOct 24, 2014I am trying to purify His-tagged protein (by Ni-NTA resin) after over-expression in BL21 cells. When i run SDS-PAGE of un-induced, induced, Lysate, supernatant, pellet and purified protein samples, the purified protein band was showing a shift to lower molecular weight than its original (where all other samples are in the same height). My protein molecular weight is ~45KDa. But after purification, i could see a band at ~30KDa. Could some one tell me the possible reason?Relevant answerMichele GalluccioOct 27, 2014AnswerThis is normal if your protein is a membrane protein.A. Rath, M. Glibowicka, V.G. Nadeau, G. Chen, C.M. DeberDetergent binding explains anomalous SDS–PAGE migration of membrane proteinsProc. Natl. Acad. Sci. USA, 106 (2009), pp. 1760–1765View20 Recommendations Gelareh Abulwerdiasked a question related to His Tag PurificationWhy don’t I see a band of my His-tagged protein in western blots?Question11 answersOct 23, 2014I have constructed a pcDNA3 vector with a gene of interest. The DNA sequence of my construct is confirmed and His tag is in frame with my protein. However, when I transfect HEK293 cells with my construct, I can t get a His tag signal in my sample using several different anti-His antibodies. However, the positive control works. Unfortunately the antibody against my protein is not specific so the only way for me to make sure that the protein is expressed is via western blotting or immunofluorescence. My protein is about 60kDa. I don’t know if the problem is with the transfection?Any suggestions would be appreciated. Thanks for your help!Relevant answerJennifer GibbonsOct 23, 2014AnswerAre you sure the transfection for the plasmid itself worked? Is transcription working? If the answer is yes to both of those, then you need to ask if the protein is being translated- maybe it s misfolded and degraded starting with the tag. Maybe it s not expressed at all.I d check all of the above before worrying about the antibody. (The antibody could still be the problem, but with little solution- maybe the antibody is sterically hindered from reaching the his tag due to protein folding). You could also stick the his tag on the other protein terminus, if that s easier....that could avoid the potential steric problem.Sounds like you have a lot of troubleshooting to do......View7 Recommendations Raju Tippanaasked a question related to His Tag PurificationWhat is the best method for purification of His-tagged recombinant human nerve growth factor?Question2 answersOct 17, 2014I tried with Ni-NTA Agarose (Qiagen) using native conditions. I also did Q-Sepharose (ion exchange), followed by Ni-NTA native conditions. Purity was not achieved in either case. Now I m trying in denatured conditions.All of your feedback is welcome.Thank you,T M P RAJU.Relevant answerRavi Kant UpadhyayOct 19, 2014AnswerImmobilized Metal Affinity Chromatography (IMAC is used to purify His tagged proteins. Mainly beaded agarose or magnetic particles can be derivatized with chelating groups to immobilize the desired metal ions, which then function as ligands for binding and purification of biomolecules of interest. Expressed His-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under specific buffer conditions. In addition, anti-His-tag antibodies are commercially available for use in assay methods involving His-tagged proteinsView14 Recommendations Roberto Arsièasked a question related to His Tag PurificationHis6 purification without NaCl buffers: is it possible?Question4 answersSep 26, 2014Hello everybody. I would like to purify proteins with a His6-tag without using NaCl in any buffer: is it possible? Do I have to change it with another salt or completely rethink the all buffer? What s your opinion?Thanks in advanceRelevant answerDominique LigerSep 26, 2014AnswerNo problem as long as your protein is stable in absence of salt...View5 Recommendations Antonio Rosatoasked a question related to His Tag PurificationCould a his-tag cut trigger the refolding of a metallothionein like protein?Question4 answersSep 22, 2014Hi to all,I have expressed and purified a 6x his tagged metallothioein like cysteine rich protein. By means of various techniques such as electrophoresis and NMR, I have realized that the protein is unfolded and aggregates. Furthermore, I have tested various experimental conditions in order to study this protein, but no significant changes have been detected.Do you think that the enzymatic his-tag removal could change something?Furthermore, this his tag is near to the cysteine rich motif, so I think that it could interfere with the folding of the protein (for example i imagine that the his tag could bind metals and bridge some cysteine), and this reason could support the his tag removal.Thank you!Relevant answerAntonio RosatoSep 24, 2014Answerok thanks to all, the tag is precisely at the c-term, and it s near to a cysteine rich motive supposed to be a heavy-matal binding domain, subsequently i m inclined to believethat the tag could interact with the motif and some metal could bridge between the residues. so i think that by digesting the tag, something could change resulting in a improving of the protein folding.View7 Recommendations Antonio Rosatoasked a question related to His Tag PurificationHow can I purify a his tagged protein with a cysteine rich domain?Question3 answersSep 15, 2014hi to all, I am currently dealing with a cysteine rich protein having a cysteine metal binding domain cxxc and another cysteine rich motif that could be a heavy metal binding domain CxxxCxxCxC. the protein is his tagged and I can t modify the sequence. the problem is that when I examine the e.coli extracts, the protein is unfolded and aggregates.any advices?Relevant answerBrian A. DowSep 16, 2014AnswerWhat part of the bacteria are you purifying it from?  i.e. Cytoplasm or periplasm?  Do you have an idea what metal it binds?  You may consider adding a periplasmic expression signal peptide to the N-terminus, such as the PelB sequence and add the suspected metal to the culture media.  The metal could provide significant stability during folding.  You can extract the periplasmic fraction by osmotic shock with lysozyme.  Also you may consider modifying your expression conditions, such as inducer concentration, temperature, and time, if you haven t done so already.View20 Recommendations Fereshteh Sadat Younesiasked a question related to His Tag PurificationWhat causes the resin of Ni-Agarose column to attach to itself and make globular objects?Question6 answersSep 5, 2014Is there any solution for removing these globular objects?Relevant answerRavi Kant UpadhyaySep 9, 2014AnswerThese are not globular objects but aggregates of agarose bound resins. You can change the pH and see no globular object will be there. Such artifacts appear only when you work in cold or near temperature of 4-8 0C. View11 Recommendations Fereshteh Sadat Younesiasked a question related to His Tag PurificationHow can PH affect binding and coming off his-tag protein and contaminant in Ni-agarose column?Question6 answersSep 5, 2014I can t understand that how PH cause his tag proteins bind to Ni-agarose resin or come off it. I want to know more about interaction. can anybody suggest a good article about that? for example in some literature decreasing PH helps remove contaminant in elution, but how?Relevant answerSteingrimur StefanssonSep 6, 2014AnswerHi Fereshteh, histidine is a unique amino acid that binds metal ions and this binding is dependent on whether it is protonated or deprotonated. Above pH 7 it is deprotonated and binds metals. Below pH 7 it becomes protonated and does not bind metals.View66 Recommendations Zhu Hanwenasked a question related to His Tag PurificationHow should I choose a buffer when I perform a GST pull-down assay? (such as salt concentration, pH?)Question4 answersSep 3, 2014Once I performed GST pull-down assay, I used the buffer as 20mM Tris.HCl, 150mM NaCl, pH 8.0. The His tagged protein was eluted by 300mM imidazole and remove the 300mM imidazole by dialysing at 4℃ overnight. But, after dialysing overnight, I found that the His tagged protein was all precipitated. So, I want to change to a buffer with higher salt concentration (such as 40mM Tris.HCl, 300mM NaCl, pH 8.0). But what I worry about is that such a buffer with high salt concentration would disrupt the protein-protein interaction and so make the result of GST pull-down assay be artificial. So, how should I solve with this problem? Thank you very muchRelevant answerPadma NanawareSep 5, 2014AnswerHi,You can dialyze your protein for a longer time (30-40hrs) with frequent changes of the buffer to remove immidazole. I have did the GST pull down assay with buffer containing 300mM NaCl and my experience says that we don t disrupt the interactions and on the contarary it adds more confidence to your experiments as you get rid of all false positive interactions. As such the washing buffer should ideally contain 300mM Nacl.BestView9 Recommendations Dilshan Gunasingheasked a question related to His Tag PurificationHas anyone used a direct immunofluorescent labelling technique for His-tagged proteins?Question4 answersAug 21, 2014I am trying to perform a localization experiment on a His-tagged protein using immunofluorescence techniques, and I ve recently got to know that there re primary anti-His tag antibodies developed with conjugated fluorophores, which eliminates the usage of a conventional secondary fluorescence antibody. But I do have doubts on how viable this is.Relevant answerDilshan GunasingheAug 27, 2014Answerthanks for the comments RalfView0 Recommendations Lewis Hunasked a question related to His Tag PurificationDoes anyone have experience with Protein Expression and Purification?Question22 answersAug 21, 2014Hi,I am new to this topic. I constructed a recombinant protein with 6 His tag at the N-terminal. The expression host is BL21 (DE3)plysS. I checked the sequence multiple times and everything is in frame. Firstly for my pilot expression, I tried to induced with 1mM IPTG at 37 degree for 1-15 hr. Then I ran it on a SDS 8-16% gel. I found the correct size protein was there but I also saw it at my 0h without IPTG. Now I have no idea how to optimize the condition and I have a break-neck deadline for this protein purification. Can I please have any suggestions? The SDS gel picture is attached. Picture1.jpg520.63 KBRelevant answerEllis C O NeillAug 21, 2014AnswerFirstly, a good, clear and concise question with the pictures to help with answering it. Depending on how much protein you need you can either spend ages optimising the production (such as using terrific broth, inducing using autoinduction media, change temperatures etc) or just scale up what you have already done and hope it gives you enough.You should not have any protein at 0hrs as  BL21 generally gives no background expression. If that is indeed your protein overlaying with a native protein, and the increase seems reasonable, then your next step is a trial purification to confirm this is correct. It is probably better to lower the growth temperature of your cells to 30C 30 mins before adding the IPTG as this helps protein solubility. Your protein expresses well after 4 hours so this is a good time to choose.For a trial purification grow a 50 ml culture and take a 1ml sample 4 hours after induction. Lyse the cells in buffer containing protease inhibitors, DNA and lysozyme and centrifuge out insoluble material. To the supernatant add around 100uL of NiNTA beads and centrifuge (gently). Wash with a buffer containing 30mM imidazole (I use 50 mM Tris pH 8.0, 500 mM NaCl). Then elute with a small volume (50uL) of 500 mM imidazole in buffer and run this on a gel. This should have purified protein if you are expressing well. Also run the insoluble fraction, the total cells and un-induced cells to check it is expressed solubly. If this goes well then you can do a large scale expression and purification with confidence. View10 Recommendations Zhu Hanwenasked a question related to His Tag PurificationHow could I elute my protein from Ni-NTA beads when do purification?Question7 answersAug 20, 2014My protein with His tag is always on the Ni-NTA beads when purification and can not be eluted.So, how could I solve my problem? Thank you for your advice~Relevant answerAmin BornadelAug 20, 2014AnswerTry pH 7.0 for the binding step. Use higher pH if the binding you achieve is poor. For washing use 300 mM NaCl and 1 mM imidazole in 50 mM phosphate buffer pH 7.0 and for elution increase the imidazole concentration in the same kind of solution to around half of the salt concentration (150 mM) or so. Then based on the result you obtain try to adjust the concentration of salt and imidazole for optimal elution. Don t forget to get rid of the imidazole in the purified protein solution before storing it.View12 Recommendations Seyed mohammad Motevalliasked a question related to His Tag PurificationHow can we separate the imidazole from a buffer?Question1 answerAug 7, 2014We have a protein in the buffer that contain Histidine amino acids. Relevant answerAnanda Ayyappan Jaguva VasudevanAug 7, 2014Answerby dialysis your protein against same buffer that lacks HistidineView0 Recommendations Adam Wei Jian Sohasked a question related to His Tag PurificationAny advice on reduced yield of purified protein upon dialysis (Pierce slide A lyzer) and concentrating (Amicon 10K)?Question10 answersAug 4, 2014Hello guys, I have recently purified a his-tagged protein via Ni beads. Based on a BSA standard, it was estimated that the eluted protein concentration was about 1 microgram per microlitre in 100 microlitres of PBS pH 8.5. However, upon dialysis (to remove imidazole and exchange buffer to Hepes pH8.5), the eventual protein concentration became 0.1 microgram per microlitre in the same final volume. The buffer exchange was performed as the purified protein will be subjected to a phosphate dependent assay and hence the need to remove PBS. However, I do not understand what might have caused the 10 fold reduction in total protein amount. The pH was carefully controlled to be away from the PI of the protein and even EDTA was added during elution to prevent potential aggregation that may occur during Ni leeching. Also, protease inhibitors were added from the start till elution and I suspect proteolysis is unlikely too. I am considering using Hepes from the start but do offer some comments on what I might have missed out. Thanks.Relevant answerAntonio ArizaAug 5, 2014AnswerHi Adam,It s a fact of life that you lose protein at every single step of purification. The worst culprits seem to be gel filtration and concentration, though gel filtration isn t as bad as people think if you take into account the loss includes all the contaminating proteins (which were certainly included in the total protein concentration calculated before loading the column).Anyway, I digress. Back to concentrators: stay away from any concentrator that uses regenerated cellulose as these are notorious for binding to many proteins. Try concentrators that have PES (polyethersulfone) membranes as they significantly reduce this non-specific binding problem. They are slightly more expensive but definitely worth the extra cost. We use Vivaspin concentrators, just make sure you choose the ones that have PES membrane as they produce concentrators with several membrane types.Another type of membrane that has been discussed in other post is nylon. According to many this is even better than PES. I have not searched extensively for these yet, so if anybody knows of a good product that uses nylon membranes, then please let me know.View6 Recommendations Robert Bronsteinasked a question related to His Tag PurificationDo you have a good protocol for DTT dialysis in TCEP?Question1 answerJul 28, 2014I am wondering if anyone has a good protocol for dialyzing out DTT in TCEP, I have some His-tagged recombinant proteins and need to get rid of the DTT for pull-downs. Thanks.Relevant answerDiego PesceJul 29, 2014AnswerHi Robert,Are you trying to avoid disulphide bond formation?I think a normal dialysis protocol under inert atmosphere should work.Good luck with your experiment,DiegoView0 Recommendations Venkat Reddy Chirasaniasked a question related to His Tag PurificationHow can I simulate beta peptides?Question4 answersJul 29, 2014Hi,I want to simulate a beta peptide flanked by chemical moieties. I have seen some articles on beta peptide simulations. Most of them have used GROMOS53A6 or 54A7 ff. So, how to generate topology information for beta peptides? Thank you Relevant answerIstvan MandityJul 29, 2014AnswerDear Venkat,We used the MMFF94x force field for the computation of beta-peptides.Best regardsIstvánView9 Recommendations Gaurav Kumarasked a question related to His Tag PurificationDoes the presence of two Shine-Dalgarno sequence affect the expression of my gene?Question6 answersJul 23, 2014My plasmid construct has two shine-dalgarno sequence. One is present 55bp upstream of gene and other is present in my primer. Does the presence of two SD affect the expression of my gene?   Relevant answerAlfonso Saera-VilaJul 23, 2014AnswerWell what I understand is that the SD helps to bind the ribosome to the mRNA near or at the ATG so translation can start. Now having two could be a problem if:1.- they are too close so the extra one can compete for the binding but if they are two far should not be a problem considering that the SD consensus is quite common (AGGAGG). 2.- if they are far enough to not compete the only problem I see is that you create an artificial translation start site by having the SD in your primer next to another ATG. If this is the case you should check your sequence and see how many extra aa you are adding, if they are in frame (33% chance), and if the new sequence is going to affect your experiment or not.Actually, reading your question again you say SD is at 55 bp upstream of your gene (I assume you mean the ATG) so it looks far to me for a SD (people say about -7/-8 from ATG).So in summary all depends on your exact sequence, and you won t know for sure until you try it. Depending on the experiment I guess you could give it a try.Good luck!View12 Recommendations Shanice Chiangasked a question related to His Tag PurificationCan anyone help with a protein expression problem in E.coli?Question11 answersJul 23, 2014I have cloned a rice gene into the pET28a His tag vector. I have confirmed recombinance by sequencing and it is in frame. I run the SDS PAGE along with my empty pET. I am attaching my gel here.Lane 1: Protein marker, 2: pellet from uninduced empty pet 3: supernatant from uninduced empty pet 4. Pellet from induced empty pet 5. Supernatant from induced empty pet 6. Pellet from uninduced pet with insert 7. Supernatant from uninduced pet with insert8. Pellet from induced pet with insert 9 supernatant from pet with insert (my protein size should be around 20kDa) 10551868_957439744282171_137161592_n.jpg271.09 KBRelevant answerAntonio ArizaJul 23, 2014AnswerHi Shanice,After a quick search online I think you might be using the XXL Deluxe marker from Geneon. If that s correct, then the bottom band (green) is 10 kDa, the next one up is 17 KDa and the next one up is 26 kDa. Your protein should be visible in between the second and the third marker bands from the bottom and it look like it either hasn t expressed at all or there is so little of it can t be seen through the native E. coli proteins.Gabriela is right and you should check if you protein is being expressed with a western blot. Since your protein is His-tagged, that should be easy enough, just use a primary anti-His-tag antibody, which will give a very strong signal even if the protein can t be seen on the gel with the naked eye.What was your expression protocol? (cells you used, temperatures, IPTG concentration, etc...)It could also be a problem with the tag. Sometimes N-terminal His-tags interfere with the correct folding of a protein for various reasons. This can generally be rectified by trying a C-terminal His-tag instead. At this stage I wouldn t try a solubilisation tag (SUMO, MBP, etc...), as these are used to make insoluble protein soluble and it doesn t look like you have insoluble protein either.If playing around with the expression protocol and the tags still doesn t work and your sequencing is definitely correct, then check for rare codons (I don t think rice should have any ... but I haven t worked with plant genes before). If it has rare codons, then choose an expression strain that codes for these codons (e.g. Rosetta 2 codes for most rare codons) or, if that still doesn t work and you re sure it is the codons that are the problem, then have the gene synthetically codon-optimised, which will produce a gene that uses the normal E. coli codons instead of the native codons from your rice gene.I hope that helped,TonyView15 Recommendations Nicolas Kylilisasked a question related to His Tag PurificationConcentrating protein using Vivaspin Concentrator takes longer than usual. Why?Question9 answersJul 14, 2014I have carried out a His-tag assisted affinity purification of a protein (43kDa) and now I am trying to buffer exchange and concentrate down my protein sample using a Vivaspin 20ml concentrator (30K MWCO) to concentrate down my protein. However it takes too long for the buffer to pass through the filter (1h-1h30min). Does anyone know whether this signifies that something is wrong with the protein sample?Thanks in advance for your time!P.S. I know I should have used a concentrator with lower MWCO but I wanted to get rid off a 27kDa protein that co-purifies with my protein.Relevant answerAntonio ArizaJul 22, 2014AnswerWe produce large amounts of protein for structural studies and they usually need to be concentrated to anywhere between 4 to 30 mg/ml. Concentration runs of one to two days are not uncommon for us, so I wouldn t worry about spending two hours concentrating your protein. We set our centrifuges to 4oC and spin them at 4000 rpm.As has already been mentioned, resuspending the protein every 15-30 mins is helpful as this stops it from forming a gradient. The protein at the bottom of the concentrator will eventually become much more concentrated than the portion at the top and it can easily crash out of solution.Also, to reduce the probability of your protein sticking to the concentrator membrane, you should choose concentrators with nylon membranes or at least PES (polyethersulfone) membranes, but stay away from any membrane that has regenerated cellulose in it.View25 Recommendations Chandni Soodasked a question related to His Tag PurificationWhat should be done when a protein is getting precipitated during dialysis?Question18 answersJul 20, 2014I purified my recombinant His tag protein in NaHPO4-50mM, Nacl-300mM,immidazole-20mM,bmercaptoethanol-10mM, glycerol-10% with pH 8 (pI of protein 9.45) and eluted in 500mM Imidazole conc. in elution buffer. I dialysed my protein for removing imidazole in PBS but it is being precipitated. What should I do to prevent precipitation?Relevant answerFrancisco J. EnguitaJul 20, 2014AnswerHi ChandniDialysis is a slow method for buffer exchange. Some proteins have time to get into weird unstable conformations and precipitate. To avoid that, I will suggest you to use a faster method for buffer exchange like PD-10 columns.Another thing is that when you dialyzed you removed two important things: ionic strength (PBS has around 150 mM of Na and K ions, but in your initial purification buffer you had 300 mM) and glycerol. I will also suggest to increase a little bit the NaCl in your final buffer and also to include some glycerol. Proteins typically need some ions to get into solution.Good luck. BestPacoView42 Recommendations Sona Chowdhuryasked a question related to His Tag PurificationOther than a linker GSGSGS do I need to add any other residues between the protein and the C terminal His tag?Question3 answersJul 17, 2014I am expressing a heterodimeric protein (baculovirus expression system) in which the transmembrane has been deleted and replaced by the his tag. I was able to detect the protein by western(denaturing) using both His and an antibody to both the proteins. I am purifying the protein which is secreted into the medium since the pH of the medium is 6.2. Then doing a buffer exchange with PBS before loading it onto the column. I have tried different binding buffers and pH, but still the protein is  not binding to either cobalt or nickel beads. I guess the tag is buried. I cannot use denaturing conditions to expose the tag hence I would like to use a linker so that the His tag is more flexible.Relevant answerSona ChowdhuryJul 17, 2014AnswerThanks for the suggestions. The reason  I do not want to put the tag on N terminal since I am expressing an heterodimeric  protein  and the proteins ineract at the N terminal.Martin I did not want to use urea since I was concerned that I will lose the smaller protein that is part of the heterodimeric protein.View0 Recommendations Fereshteh Sadat Younesiasked a question related to His Tag PurificationIs there any difference between pure and impure enzyme for determining the PH and temperature optimum?Question7 answersJul 12, 2014I need PH optimum for enzyme for doing a good dialysis, so I determined PH and temperature optimum by impure enzyme. Is there significant difference with pure enzyme?Relevant answerMichael J. BenedikJul 13, 2014AnswerI have to disagree, the temperature and pH optimum of the enzyme, whether pure or crude, will be the same. However exogenous factors could affect the observed optimum. For example if your extract has a protease in it that is activated at some temperature then you would lose activity at that temperature. It would still be the optimum for your enzyme, just not the optimum for THAT ONE PREPARATION of the enzyme.  But in general I would expect the two measurements to be fairly close, assuming your crude extract is reasonably stable.View24 Recommendations Prabhadevi Venkataramaniasked a question related to His Tag PurificationHow can I solve my protein instability so it can be used in NMR and ITC studies?Question13 answersJul 9, 2014I have a highly charged low molecular weight bacterial protein that is highly unstable. I have tried purifying it with different tags including His tag, MBP tag, Thioredoxin tag, SUMO tag and GST tag. Of all tags used, the protein was found to be the most stable with the GST tag. However, if I cleave the tag, the protein becomes unstable and unsuitable to be used in NMR and ITC studies. The N-terminal His tag makes the protein highly unstable. Im looking for purifying this protein with a small tag. Presently, I am trying to add 5 lysine residues after the N-terminal His tag to purify my protein. Could you offer me a few suggestions? ThanksRelevant answerAntonio ArizaJul 9, 2014AnswerHave you checked the isoelectric point of your protein? If the protein has a PI of 8 and your buffer is pH 8 then the protein won t be charged properly and therefore crash out of solution quite quickly. Always choose a buffer system that is about 2 points away from the PI of the protein (e.g. for PI = 8, choose MES, BIS-TRIS or BIS-TRIS propane, all of which will buffer around pH = 6).If an N-terminal His-tag doesn t work, maybe the problem lies with the position of your protein s N-terminus, which might be somehow disrupted by the tag or the tag interacts/occludes the active site. In these cases changing to a C-terminal His-tag often works.As Helge and Justin already mentioned, highly charged proteins usually need buffers with high ionic strength. For structural biology we tend to keep our proteins in buffers that have very low NaCl concentrations as NaCl (which is a chaotroph) can inhibit crystallisation, but if the protein needs 200 or 300 mM NaCl to stay in solution, then you should at least try and see if your experiments still work with such a buffer. If this is not an option you should investigate other buffer systems like the argenine/glutamate mix mentioned by Justin. View15 Recommendations Yin-biao Wangasked a question related to His Tag PurificationCan anyone help me with Ni-NTA affinity purification?Question4 answersJul 7, 2014Dear All,A large amount of soluble protein of my interest could be harvested in E. coli. Yet, the protein seems to have rather low binding capacity with Ni-NTA column. The addition of 20 mM imidazole in washing buffer catalyze the elution of my protein together with other proteins.   Both 50 mM Tri-Hcl and 50 mM NaH2PO4 have been tried for using as the buffering conditions. pH 7.4 and pH 8.0 have also been attempted, so did the concentration of NaCl. To avoid the possibility that the His-tag might have been masked, I tried to induce my protein into inclusion bodies and purified it in 8 M urea. The same problem appeared. Even under denatured conditions, 20 mM imidazole lead to the elution of my protein from the column. Then, I tried to modify my protein by adding two more His tags on its carboxyl end. This newly expressed protein was expressed as inclusion bodies, and it shows a higher binding capacity with Ni-NTA. 30 mM imidazole result in its elution from the column with other proteins. So my problem here is that Ni-NTA affinity chromatography seems not to be able to be used as a method for purifying my target protein. Can anyone give me some help on affinity purification of His-tagged protein?Relevant answerSimon GebremeskelJul 7, 2014AnswerHi, I had a similar problem and the following youtube video was a huge help and solved my problem. I think the amount of imidazole you are using is too low, esp the 20mM concentration. I would suggest doing a step-wise elution with increasing concentration of immidazole, this allows you to was away any contaminating proteins with the lower concentration of imidazole but get your protein of interest at the high concentration of imidazole.  I think the youtube video below gives an excellent starting point on how to fix yoru problem. I hope this helps. https://www.youtube.com/watch?v=y0ffJBFQswkView21 Recommendations H P Dulalasked a question related to His Tag PurificationHow can I solubilize an inclusion body?Question2 answersJul 7, 2014I am trying to purify His-tag Dectin-1 protein as an inclusion body. After I sonicate the dectin-1 expressed E. Coli cells I centrifuge and wash with phosphate buffer. For the solubilization of inclusion body, I am using 8M Urea solution but it is not solubilizing completely and looks turbid. Do you have any suggestion for the proper solubilization of inclusion body? Relevant answerIngmar StrobelJul 7, 2014AnswerHi Hari,I would also recommend to solubilize the lysate with GuHCl (6-7M). It is important that the solution is not too cold. Bring the mixture to 25-30°C in a water bath, thus it will help that the solubilization becomes more complete. View0 Recommendations Maciej Zacharskiasked a question related to His Tag PurificationHow to reduce the haemoglobin contamination after his-tagged protein purification on cobalt resin?Question2 answersJul 2, 2014Do you know any procedure to reduce the amount of haemoglobin which is co-purified with the protein of interest during cobalt affinity chromatography? We ve just started to use the Promega reticulocyte lysate kit as a cell free protein expression system. After translation we have both the protein of interest (13 kDa) and haemoglobin. Unfortunatelly, after purification, we got mostly haemoglobin, instead of the protein, we were expecting. Promega s advice is to use their MagZ™ Protein Purification System, however I would like to avoid it. Any ideas?Relevant answerVivica GrotelueschenJul 3, 2014AnswerI also worked with the reticulocyte lysate a while ago. First of of we did a qick spin of the lysate before using the supernatant only.To get rid of hemoglobin we did an ammonium sulfate precipitation.View6 Recommendations Dhaval Patelasked a question related to His Tag PurificationAfter purifying my protein it gets easily degraded when I am concentrating it. What would be the solution?Question15 answersJul 1, 2014My protein of interest is actually of 39kDa and after purification I am getting at 66kDa and through MS/MS analysis it is proven that the band is actually the same 39Kda protein. My lysis buffer includes 50mM TRIS, 200mM NaCl, 10% glycerol, 1mM PMSF, 0.1% triton X. After purification when I am dialysing or concentrating (with sucrose and centricon) it get degraded. Can anyone please suggest me the reason and solution?Relevant answerAlejandro MartinJul 1, 2014AnswerThose gels you showed, are they reducing or non-reducing SDS-PAGE? Does your protein have cys residues? Have you done Western blotting to ensure that those bands appearing after concentration are degradation products? Is your protein a protease?View6 Recommendations Michael Reiterasked a question related to His Tag PurificationWhy do I have an unknown His-tagged protein?Question17 answersJun 14, 2014I am producing two proteins with E. Coli. One is fused to a his-tag, the other one to a Thioredoxin tag. Both can be cleaved off with TEV-protease. When I run a gel after purification, I can detect both my proteins, but also a third protein of unknown origin. This means however, that it is somehow his-tagged and the tag also gets cleaved off by TEV-protease.Do you have any suggestions as to what the reasons for this are?Relevant answerMario SchubertJun 16, 2014AnswerThere are a couple of proteins that stick to Ni-NTA and can co-purify. Some known proteins are YodA, Hfq and FKBP. For YodA co-purification please see Tian et al 2005, Meth. Enzymol. 394, 321-334.View42 Recommendations Leonardo Guzmanasked a question related to His Tag PurificationProblems with the elution of His-tag protein - Can anyone help?Question4 answersMay 22, 2014We are trying to purify the G beta gamma dimer with the His6 tag technique. We obtained pretty good levels of expression, but the protein is completely retained in the resin. We have tried eluting with 250 and 500 mM imidazol, but the elution is very low. I would be greatful for any suggestions.Relevant answerLeonardo GuzmanJun 11, 2014AnswerThanks so muchView0 Recommendations Maximilian Paradizasked a question related to His Tag PurificationWhat method can I use for purification of a His-tagged synthetic peptide?Question4 answersJun 5, 2014We want to order a 20-mer synthetic peptide. We are considering adding a His-tag to the 20-mer (making it a 26-mer) and ordering the crude product, and then purifying it ourselves using his-tag affinity column. Have any of you tried this and had any positive or negative experiences, or knows of any reason why this is would be a bad idea?Relevant answerMichael SwansonJun 5, 2014AnswerDo they tell you how pure the synthetic peptide will be? I think the problem with adding a His-tag might be the fact that any shorter/longer product impurities from synthesis will also have the same his-tag, and thus you will not be able to separate the impurities by affinity column. I do not have any specific experience with this though. I am interested hearing more opinions.View20 Recommendations Nafiseh Sanei ata-abadiasked a question related to His Tag PurificationDoes anyone have experience with purification of secreted his tagged proteins from a condition medium OF HEK293T cells?Question4 answersMay 28, 2014I want to purify my secreted his tagged protein from condition medium of HEK293T cells using HIS SELECT AFFINITY GEL OF SIGMA - BATCH method (NOT column chromatography). Does anyone use this method? I want a good protocol for it.Relevant answerNafiseh Sanei ata-abadiMay 30, 2014AnswerEric thanks for your suggestion.View0 Recommendations Fereshteh Sadat Younesiasked a question related to His Tag PurificationWhy did the protein aggregate after dialysis?Question20 answersMay 26, 2014I purified my protein with 250 mM imidazole, after 2 days, I remove imidazole with dialysis, but I can see a white pellet in protein.My protein is an enzyme, it has activity immediately after purification, but after 2 days, it doesn t have activity after and before dialysisRelevant answerChristopher ToselandMay 27, 2014AnswerI have frequently found precipitant when dialyzing imidazole out of the buffer. Very little of my protein was in the precipitant therefore it is usually just the imidazole itself. Have you checked if you loose protein during the dialysis step?As for the enzyme activity, it could be that keeping the protein in imidazole buffer for two days harms the protein. Also, how do you keep the protein for those two days? It would probably be best to complete the purification as quick as possible then store the enzyme in a more suitable buffer and get it frozen away.View24 Recommendations Pascal Fenderasked a question related to His Tag PurificationHow can I detect expression and detection of his-tagged Cadherin domains in a HEK293 cell?Question3 answersMay 12, 2014We express different constructs of cadherin domains from desmoglein-2 (DSG-2) carrying either one cadh domain, two or four cadh domains. All the constructs have a His-tag in C-ter and are expressed in HEK293 cells by transfection. We are unable to see the protein (cell or supernatant) by western blot using anti-His antibody. However we have checked by immunofluorescence using anti-his labelled with a fluorescent dye and the protein is really expressed in the cytoplasm. Any of you have already encountered this kind of problem with cadherin expression? Any idea that would help?Relevant answerSamantha StehbensMay 14, 2014AnswerSome antibodies work well under native conditions (IF), then fail in denatured applications (western blot). Find another few His-tagged proteins - express them, make lysates. These will tell you if you antibody works for westerns. Alternatively- look for the antibody you use in publications for western blotting- can there get it to work? Finally- you may need multiple tags- His is pretty small from memory....View7 Recommendations Zaheer Ahmedasked a question related to His Tag PurificationWhy is my HIS tagged peptide-tomato recombinant protein not released from nickle beads in the column after adding elution buffer?Question11 answersMay 9, 2014The other fusion proteins are eluted well. But there is something wrong with only one protein. It is expressed well in 500 ml LB with 0.2mM IPTG, overnight incubation at 20C and 150rpm. The problem is elution. The elution buffer contains 500mM imidazole and pH is 6.0. I am looking for a solution and some recommendations.Relevant answerEmir Salas SarduyMay 12, 2014Answer50-100 mM EDTA is used to remove all the metal ions from the resin in stripping protocols, so these conditions can be used to elute your protein. EDTA can then be removed from your sample protein by PD10 or dialysis. Just remeber to recharge your column with fresh metal to re-use it.View6 Recommendations Min Han Lewasked a question related to His Tag PurificationWhat strategy is better for elution during Ni-NTA affinity purification, imidazole or pH gradient?Question8 answersMay 9, 2014Imidazole and pH gradient are the two most common strategies used in the Ni-NTA purification of his-tagged recombinant proteins. Imidazole is the competitive inhibitor for histidine-tagged protein, while the pH gradient offers elution of protonated his-tagged recombinant protein from basic (pH 7-8) to acidic condition (pH 4-5). To my understanding, imidazole and pH gradient can both be used under either native or denaturing conditions. The question is, in commercial protocols, why is the use of imidazole always preferred in native purification? And also why is pH gradient always used in denaturing purification?Relevant answerEllis C O NeillMay 9, 2014AnswerImidazole can also be used across a gradient to obtain purer protein. Proteins will be more stable and more active at the neutral pH of imidazole purification (unless inhibited by imidazole). Proteins are generally less stable and less active at low pH. Those that are stable may not be stable at the starting pH (8.0). I would recommend using imidazole - make sure you use a high purity supply which does not absorb at 280 nm for monitoring by UV absorption.View35 Recommendations Álvaro González Martínezasked a question related to His Tag PurificationWhat is the best method for separating and isolating a purified protein from the His-Tag in a solution?Question3 answersMay 8, 2014We purified a protein marked with His-Tag and with a TEV cleavage site. We would like to know ideas about how we can separate the mix of proteins in the solution after the addition of TEV protease.Relevant answerAlejandro MartinMay 8, 2014AnswerTEV protease itself is usually His-tagged, so you run the mix through an IMAC column and get pure cleaved protein in the flowthrough.View20 Recommendations Mariam Mahmoudasked a question related to His Tag PurificationIs dialysis required before purification of ammonium sulfate precipitated His tagged proteins using Ni-NTA columns?Question4 answersMay 7, 2014See aboveRelevant answerEllis C O NeillMay 7, 2014AnswerIt is not strictly necessary. Your sample must be completely solublised to prevent clogging the column. Also the high ammonium sulphate may encourage your protein to stick to the column s hydrophobic material. However as IMAC is a concentrating technique you can dilute your protein down to 100mM ammonium sulphate (everything will be soluble) and then pass your whole volume over the column - your protein will all stick and be eluted in one step.View4 Recommendations Dhaval Patelasked a question related to His Tag PurificationMy protein of interest is of 39kDa and after purification with HIS tag, I am getting a single band at 60kDa. Why?Question35 answersApr 23, 2014I have cloned a gene in pET15b, pET28a, pET30a and used BL21, Rossetta and pLysS cells for expression but after purification I get a single band at 60kDA . I have added BME in loading dye, so the problem of a dimer is not in the case. Sequencing is also in frame and correct. One thing I would like to add is that my protein has a +ve charged a.a. tail. (last few residues - RRKKKMK). Also attached the picture of the tag purification. IMG_1427.jpg605.03 KBRelevant answerA researcherApr 23, 2014AnswerAlthough it is believed SDS-PAGE should resolve proteins according to their mass, I also has some proteins which run at much different hight to what I would expect (largest shift I saw was 20 kDa for 20 kDa protein, or 30 kDa for 100 kDa protein, it was running as 70kDa). And I am sure I am not the only one observing it. If you have His-tag, you could try to detect it with Western-blot.Also sending your sample for MS would be an option. If your protein can bind RNA (you have plenty of tRNA in Rosetta) or DNA, although they should be separated on this kind of gel, it may happen they still run together (I saw it also for very strong interaction). As a control, you could treat your sample with RNaseA or DnaseI.View42 Recommendations Brian Broom-Peltzasked a question related to His Tag PurificationWhat are different ways of purifying heterodimers?Question2 answersApr 17, 2014I am attempting to purify a malate thiokinase (E.C. 6.2.1.9) recombinant heterodimer from Methylobacterium extorquens. The two sequences are currently expressed on the same plasmid and promoter with one of the sequences expressing a HisTag. During the first expression attempt, I was only able to purify a minimal amount of protein. I have looked up different methods, including expressing the proteins separately and denaturing them with urea and refolding them by dialysis and was hoping to get some recommendations on different methods. The original methods used by Hersch to purify the protein used protamine sulfate precipitation and two chromatography steps. I am hoping to be able to still use the HisTag.http://www.jbc.org/content/248/21/7295.longRelevant answerJens Mark MollApr 18, 2014AnswerOne way to do this could be expressing the two proteins as a fusion protein by connecting the two proteins with a flexible linker (e.g. GGGGS x X). This guaranties equal expression levels of both parts of the protein. Of course there are limitations to such an approach including sterical problems due to the linker sequence. If there is a crystal structure available you can check if it is likely possible to use a fusion protein approach.The Fusion protein can then be easily purified via your His-tag. Activity assays along the way will ensure the protein is functional as a fusion protein.View6 Recommendations Yijun Zhuasked a question related to His Tag PurificationCan someone advise on the his-tag cleavage analysis?Question12 answersApr 11, 2014As my protein is fairly big, about 80kDa, SDS-Page does not really help to show show his-tag cleavage. Is there any other way to analyse cleavage?Relevant answerAlexander TischerApr 11, 2014AnswerThere are antibodies available directed against His-Tags (http://www.abcam.com/6x-his-tag-antibody-hish8-ab18184.html). Or you could give it to someone that can do mass spectrometry. Alternatively I would just try and see if any of your protein molecules still stick to Ni-NTA Sepharose. You could do that in a test tube. Just mix your protein with some matrix, spin the matrix down, wash it several times with some imidazole and then elute with more imidazole (Wash- and Elution buffer should have the same Imidazole concentratrion as whatever you use for purification). Then do a TCA-precipitation of the eluted protein and load it on an SDS-Gel. When you get no protein on that gel - there are no His-tagged species in your preparation. The positive control for this assay would be to do the same with some non-cleaved protein and see if it works.Cheers.View20 Recommendations Mariela Luján Tomazicasked a question related to His Tag PurificationHas anyone tried HisDetector Nickel-NTA for WB analysis of polyHis-tag proteins?Question4 answersApr 2, 2014I have performed the purification of polyHis tag proteins in IMAC and eluted with high concentration of imidazole. Is it necessary to remove imidazole prior SDS-WB when using His Detector Nickel- NTA?Relevant answerMariela Luján TomazicApr 4, 2014AnswerThanks to all for your answers. Yes, I know that there is no need to remove imidazole in standar WBs that use antibodies. But in this case I am using a Nickel reactive (HisDetector Nickel-HRP, KPL) that compites with imidazole for histidines. That´s why I am asking if anyone has tried before. Thanks again!View0 Recommendations Ricky Laliasked a question related to His Tag PurificationWhy does the second elution fraction (out of 3 total) contain the greatest amount of target protein?Question2 answersApr 3, 2014I ran a hexa-his tagged protein through Ni-NTA column and used elution buffer (250mM imidazole) to elute the purified product. After running on SDS PAGE, I found that the second elution fraction contained the highest intensity band. Is there an explanation for this?Relevant answerPrem SubramaniamApr 3, 2014AnswerAs a rule of thumb, when a protein BEGINS eluting - that is, the buffer conditions are right for complete unbinding from the matrix - the first hint of protein will occur ONLY after the dead volume. Dead Volume = Assume a protein does not bind the column at all, or is completely begun unbinding the matrix. It will take time for it to travel from the top of the column to the bottom. The volume of buffer required for this is the column dead volume . Any protein eluting before the dead volume would indicate the column is faulty. Thus, when you load your CRUDE extract FOR THE FIRST TIME, the first fraction of the flow through that contains protein will determine the dead volume. Again, rule of thumb dead volume is about 30-35% of bed volume. Elution Volume = Volume required to COMPLETELY elute protein and is usually = 2 x bed volume, ASSUMING the protein is completely eluting with hat buffer. So it is the sum of dead volume + volume of fractions that contain protein. So your first fraction, at the time of adding elution buffer should NOT contain any protein, because it is the dead volume. If your fraction volume is dead volume, but elution volume the first fraction will have less protein than the next fraction. Let us assume you collected 3 fractions of 1 ml each for the above expt you mention. Now if you had collected 6 fractions of 0.5 mL each, then your protein will be maximum in between fractions 3 4. It is a VOLUME thing and not a fraction number thing. The more fractions you collect for the same total elution volume means you have to collect less and less volume per fraction.There is an advantage is stretching out fractions with smaller volume size. This allows, for finer separation FRACTION wise (or volume wise) between two proteins that elute in very close. Thus, if you elute for 1 ml, if protein A elutes in 0.5 mL and protein B immediately following it, then using two 0.5 ml fractions gives you a better chance of separating A from B in different fractions. If you collected all 1 ml in same fraction, A will be contaminated with B.Think this through, it is not that difficult !! :-)View4 Recommendations Yavuz S Dagdasasked a question related to His Tag PurificationDoes high concentration of imidazole(~1M) affect protein-protein interactions?Question8 answersMar 3, 2014I am co-expressing two protein, one having 6x-His tag and other without any tag. When I do protein purification I have used 1M imidazole for elution. Since my prep was not clean enough I have decreased the Imidazole concentration and repeated the Ni-NTA purification again. Now I have a clean elution but the second protein that is not tagged is lost. Is it possible that 1M imidazole affects protein-protein interactions?Relevant answerOleg LaptenkoMar 4, 2014AnswerHi there,In addition to competing with your His tagged protein in the metal affinity chromatography, imidazole also behaves just like any salt: means it may interfere with any protein-protein interactions that are mediated by polar or charged residues (ionic interactions). Therefore, 1M imidazol is way too much. Usual range of imidazole in the elution buffer is from 50-250 mM and that will dependent on the overall ratio between the amount of your tagged protein and number of the affinity sites available for the interactions (means, amount of the Ni resin). One more thing. As Tamjeed has mentioned above you need to pH the elution buffer that contains imidazol, as on its own imidazole is very basic, which may be an extra factor responsible for loss of protein interactions in your assay.Good luck.View28 Recommendations Xiaowen Xuasked a question related to His Tag PurificationCan someone recommend a good anti-his tag antibody for western blotting?Question5 answersFeb 20, 2014Most of my proteins are his-tagged (6x). I m shopping for a robust antibody to exam these protein with western blotting.Relevant answerNirajkumar R. MakadiyaFeb 20, 2014AnswerHi,Thank you for raising this very important question. Recently, I tried to detect the bacterially expressed His-tagged protein with anti-His antibody and the experience was far from good. 1.) Anti-His antibodies are known for their specificity and preferences and notoriety. Anti-His antibodies are very much sequence dependent. 2.) Qiagen sells Anti·His Antibody Selector Kit which has 3 antibodies in it. 3.) If possible use a positive control for western blotting.4.) Read this article Debeljak N, Feldman L, Davis KL, Komel R, Sytkowski AJ. Variability in the immunodetection of His-tagged recombinant proteins. Anal Biochem. 2006 Dec 15;359(2):216-23. Epub 2006 Nov 1. PubMed PMID: 17081490; PubMed Central PMCID:PMC1821093.Good luck!View18 Recommendations Basem M. Ahmedasked a question related to His Tag Purification6His tag introduction into a non tagged vector?Question11 answersFeb 15, 2014I am going to clone some genes in an expression vector, as purification is needed in the work flow, I thought about 6His tag and Ni resin purification but the vector itself is non tagged.2 sequences encode the 6His tag:5 -CAT CAC CAT CAC CAT CAC-3 5 -CAT CAT CAC CAC CAC CAT-3 which will be better to be added downstream the reverse primer sequence?And what is the possibility to hit a positive PCR from the 1st time using the tagged reverse primer which may be 40 base or more long?Relevant answerKyle H RohdeFeb 16, 2014AnswerFor scenarios like this, we have found a PCR method called Round the Horn PCR (http://openwetware.org/wiki/%27Round-the-horn_site-directed_mutagenesis) to be very useful. Essentially, you order primers to amplify around the whole vector, including your inserted sequence (His tag plus cleavage) on the 5 end of the forward primer. Prior to PCR, you phosphorylate the primers with T4 kinase and then circularize the linear product by ligation before transformation into E. coli. We have the best luck with polymerases like Phusion or Q5. If your gene of interest is already in the desired expression vector, this way can avoid unnecessary cloning steps. If interested to try this way, I can provide more details if necessary.View18 Recommendations Srividhya Sundaramasked a question related to His Tag PurificationDoes His tag affect enzyme activity?Question5 answersJan 30, 2014I am working on an enzyme (42 kDa) with N-His tag, purified from E.coli BL21 codon plus. Upon checking for activity, the enzyme is highly active, but when the His tag is removed, the activity is lost. We are performing the assay with Tris buffer at pH 8, both for His/-His enzymes. Any suggestions for this?Relevant answerAlejandro MartinJan 31, 2014AnswerYes, there is always the theoretical possibility of His tags affecting quaternary structure (there s even a couple of real life examples in Carson et al, His-tag impact on structure, PMID 17327666), but what I find amazing in your case is that the tag is apparently required for enzyme activity. Are you expressing a full-length enzyme or a truncated variant? If the latter were true, perhaps the His tag is serendipitously fullfiling the role of the missing part in the original. Or perhaps the natural enzyme is active only as an oligomer, or associated to an interacting partner? His tags are known to mediate oligomerization via metal cations, so...honestly, I m out of ideas here.View5 Recommendations John Tainerasked a question related to His Tag PurificationWhat would you recommend as the best tags for large-scale purification of proteins?Question44 answersDec 1, 2013We wish to purify proteins that may be modified and in complexes in as few steps as possible for structural studies.Relevant answerTom HoppDec 3, 2013AnswerI invented the FLAG tag, so perhaps I m biased. Nevertheless, there are unique benefits. The flag sequence is much more hydrophilic than his6, so it does not tend to insolubilize the protein. While antibody affinity columns are expensive compared to nickel columns, there is another factor. I have used an anti-flag-M1 monoclonal affinity resin repeatedly without degradation of the binding ability (12 runs with the same column, still ~100% binding capacity. This is because the entire purification can be carried out at pH 7 in saline, and no acid elution is needed. Flagged proteins bind to the M1 column in PBS + Ca and release from the column in PBS + EDTA. This is VERY mild treatment for the protein, and the column. Note: must be anti-flag M1, not M2 or M5. Before and after purification, the monoclonals can be used to follow the protein (M2 is best for this), isolate it when complexed to other proteins (M1 is best), and western blot the protein (M2). So you get a lot of tools with the FLAG toolkit.View60 Recommendations Fabian Fischerasked a question related to His Tag PurificationPeculiar tandem affinity (6x His-TEV-Strep tag II) purification problem - any suggestions?Question26 answersJul 17, 2013My aim is to purify an oligomer-forming protein from isolated fungal mitochondria by TAP. For this purpose I have appended a C-terminal 6x His-TEV-Strep tag II. It should be noted that switching the tag to the N-terminus is, for various reasons, not possible.With a different monomeric mitochondrial protein (\"control protein”) with the exact same tag TAP can be done without a problem. For my protein of interest (POI) I have verified by Western Blot that the full length tag is present and not degraded during lysis/binding steps. Native purification with the 6x His tag works just fine, so supposedly the tag is accessible and not folded away inside the protein. However, there is absolutely no binding of my POI to StrepTactin either in column (StrepTactin Superflow Columns, IBA) or batch (Qiagen StrepTactin Magnetic beads) procedure using the following buffer for lysis/binding: 0,5 % Triton X-100, 300 mM NaCl, 20 mM KPi (pH 7,4) and 1 mM PMSF. The same buffer works for 6x His purification of my POI and also for both 6x His and Strep tag II purification when I do it with the control protein. So I assume there is no general problem with buffer components/composition/handling. In theory I can see no reason why the Strep tag II purification should not work (tag is present, stable and presumably accessible) so I’m really at a loss.1) Has anyone ever encountered a similar problem, where of two consecutive tags only one was accessible for native purification? I guess it depends on the respective protein, but is it actually feasible that the Strep tag II is sterically inaccessible even though the preceding 6x His tag isn’t?2) Is it possible for the Strep tag II to somehow become modified and thus unable to bind to StrepTactin?3) Any suggestions for additives/alterations to the buffer to enhance binding?4) Would it be advisable to place a Twin-Strep tag directly at the C-terminus seeing as the 6x His directly at the C-terminus is definitely accessible and as this would probably reduce background?5) Any suggestions for other tags that can be combined with the 6x His tag for TAP from mitochondria?Relevant answerFabian FischerNov 13, 2013AnswerJust FYI, if anyone is still reading this topic: I tried different tag variants with my POI, including a Twin-Strep-tag and a 3XFLAG-6xHis-tag (+/- Poly-GGGGS-Linker) in the same C-terminal position as before and purification with these variants worked reasonably well already on first try, though I can t yet tell wether the tags interfere with POI function. In any case, as some have commented on this being possibly a hindrance for purification of Strep II-tagged proteins, presence of up to 1 % Triton-X-100 in the lysis-/binding-buffer is no problem for Twin-Strep-tag purification in my hands. My best guess for Strep-tag II purificiation not working with the inital construct described above (6xHis-TEV-Strep-tag II) is still that there is a problem with sterical accessibility of the tag in this particular context - then again, if it doesn t work you never know why.Again, thank you all for your helpful suggestions and comments.View20 Recommendations Priya Aroraasked a question related to His Tag PurificationHow to check the concentration of protein per litre of culture?Question3 answersOct 19, 2013I have checked the concentration of my protein using Bradford method but I want to know its concentration per litre of culture.Relevant answerMaulik RachhOct 22, 2013AnswerDid you mean Purified protein concentration by Bradford or protein in crud form..?View0 Recommendations Ajeet Singhasked a question related to His Tag PurificationWhat to do when protein is getting precipitated during dialysis?Question22 answersOct 4, 2013I purified my recombinant His tag protein in Tris, pH 7.5 (pI of protein 8.9) using 400mM Imidazole conc. in elution buffer. I dialysed my protein for removing imidazole but it is being precipitated. What should I do in such kind situations?Relevant answerJohn TainerOct 10, 2013AnswerFirst I would put a drop of the precipitate solution on a glass slide and look at it under a microscope – is it crystalline or amorphous? If it is crystalline you may be able to spin out a highly purified sample of your protein and re-solubilize it. If it is not crystals, you can adjust the bottom lighting you can easily see precipitate as a cloudy drop and try pushing the pH, salt or other additives up or down by pulling a liquid thread connecting with drops of different solutions. In this way you can test with a drop if any changes will re-solubilize your sample. Once you know what may help, you could switch buffers quickly by running your sample down a de-salting column instead of dialyzing, which is slow.View80 Recommendations Muhammad Jbaraasked a question related to His Tag PurificationWhy hasn t His Tag protein eluted from Nickel column?Question9 answersOct 9, 2013I have tried to elute His tag protein (37AA) from the Ni column by 200mM imidazole buffer for 30 minutes, but I obtained only a small percent of His tag protein that eluted. Does anyone have any advice as to how to do this more efficiently and in less time?Relevant answerRebecca RostonOct 10, 2013AnswerAll the answers so far have good ideas. Stripping the ions from the column using excess imidazole (I use 250 mM standard for elutions, btw), EDTA, pH, etc will probably remove any protein that remains.However, I would start by making sure that your protein is still attached to the column. Did you check your starting material and unbound fractions? A common problem with His-tags is that if they are imperfectly exposed (partially stuck inside a folded protein), they do not bind well. If it is possible that your current elution problems are actually binding problems, then none of the above strategies will work. If the problem is actually binding to the Ni, it is possible to increase the specificity of binding to Ni by using denaturing conditions, or gentle detergents, depending on the protein. If the problem is actually elution, I would recommend trying 20 column volumes of 250 mM imidazole, and collecting these in testable fractions. I have occasionally purified proteins that seem to bind Ni exceptionally well, and have come off in elutions 12 - 15. Then I would try strategies that strip the Ni, like EDTA and pH. You can find excellent recommendations in the freely downloadable Qiaexpressionist manual from Qiagen.Good luck!View16 Recommendations Ehud Shaharasked a question related to His Tag PurificationCan anyone explain results of an protein detection experiment?Question4 answersOct 9, 2013These are the results of an old experiment, which I still find hard to explain.In cloned proteins with 6xHis, sometimes one can t detect 6xHis in the folded form, since it is buried in structure and might be unavailable for detection antibodies. After denaturation we can usually detect protein through 6xHis with western blots.In our lab a colleague of mine cloned a protein, and couldn t detect it with the same anti 6xHis antibody in western blot, although a band in the right size appeared strong in coomassie staining (checked vs. 0 time of expression in production process). After performing ELISA or Dot blot we could see good staining. Protein was detected in the folded form, and not denatured, eventually it was also shown to be present in samples.Can anyone offer an explanation?Relevant answerMarcia MossOct 9, 2013AnswerMaybe most of the protein does not have the His tag, but a small population still does, so it is a mixed population of proteins. Since the Elisa is more sensitive, it is detecting the small population of proteins that still has the His tag.View10 Recommendations Lucas F. Ribeiroasked a question related to His Tag PurificationCan a 6xHis tag bind streptavidin?Question4 answersOct 3, 2013Did anyone have this problem?Relevant answerEhud ShaharOct 3, 2013AnswerLike Younis and Paul said it should not bind.There is an enzyme called biotinylase found in coli (if this is your expression system) may be your protein has biotinylation motive and was biotinylated. Interesting enough, you can find biotinylated proteins in humans as well so probably we have an equivalent.Long shot but it is worth checking.View3 Recommendations Shibi Mathewasked a question related to His Tag PurificationElution of his tagged protein?Question4 answersSep 23, 2013I know for sure that my protein is his tagged . I know that the protein is binding to the column. I am using the his trap (Hp) 1ml column from GE. My elution buffer has 20mM sodium phosphate 500mM imidazole 500mM sodiumchloride . I use a immidazole gradient in the elution and there are no elution peaks at all.Relevant answerShibi MathewSep 27, 2013Answerthe uv trace does not rise and the flow through also does not have the protein . I know that the protein binds because i incubated the protein lysate with the slurry and ran a sds page . I was able to get the an elution peak at 450mM only once but unable to replicate the result all other subsequent injections after the flow through there is no rise in the UV .View0 Recommendations Amruta Mhashilkarasked a question related to His Tag PurificationShould I refold my his-tagged protein after Triton-X100 digestion?Question18 answersAug 12, 2013I am working on ligand binding domains and I am expressing the protein in the yeast system. I have solubilized my protein using Triton-X100. I do know that it is a non-ionic detergent and it should not denature the protein. But, yet should I still attempt to refold it? Does Triton-X 100 have any deleterious effect on protein folding? Can anyone suggest more literature on the same?Relevant answerMuralidhar ReddivariAug 12, 2013AnswerAmrutathe answer for your question is No.. you dont need to refold protein.Triton -x -100 is a classical example of a non-ionic detergent. The nonionic detergents differ with the ionic ones in that their headgroup is uncharged and hydrophilic. They are considered mild surfactants as they break protein-lipid, lipid-lipid but not protein-protein interactions and most of them do not denature proteins. Therefore, proteins are solubilized and isolated in their native and active form retaining their protein interactors. This is their main advantage and preferred in isolation of membrane proteins. Triton X-100 is a non-ionic detergent used to denature cell membranes without denaturing the protein. Triton X-100 breaks up protein aggregates to promote enzymatic activity. information sourced and complied from multiple papers / vendorsView40 Recommendations Ciara Christianne Limasked a question related to His Tag PurificationWhat if the elution buffer for protein purification turns yellow?Question5 answersAug 12, 2013I prepared a buffer containing the following: 50mM sodium phosphate, 250mM imidazole, 3M NaCl and 50% glycerol. When I autoclaved them all together, the solution turned yellowish. Is it still okay to use? I haven t done this before. (The product manual for the resin says that these concentrations with the glycerol should work just fine... but I m not sure.) Thanks!Relevant answerSagar SridharaAug 12, 2013AnswerThe following website quotes the following about imidazole-containing solutions: http://www.applichem.com/en/shop/product-detail/as/imidazol-pufferqualitaet/ Solutions of imidazole are stable for several years ( at least 6 years). It is not necessary to autoclave the solution and to store it protected from light. Buffers containing imidazole will turn to yellow. These solutions are still suitable to elute His-tag proteins View15 Recommendations Moumita Basuasked a question related to His Tag PurificationWhat s the most efficient method to purify His tagged protein from inclusion bodies without using HPLC?Question2 answersJul 4, 2013BL21 cellsRelevant answerMary HamiltonJul 8, 2013AnswerImmobilized metal chromatography is supposed to be the best method. I do not use IMAC for that purpose because I am not expressing a His-labeled protein. BUT I am trying to separate some metallo enzyme isoforms that differ in pI.View1 Recommendation Saaraj Guptaasked a question related to His Tag PurificationSelectively visualize his-tag fusion proteinsQuestion4 answersMay 20, 2013I need to know if there s a way I can specifically quantify (visually) his-tag fusion protein. I have checked for commercial his-tag stain reagents, but I m looking for state-of-art method(s). Please send your suggestions.Relevant answerNor Aziyah MatRahimMay 27, 2013AnswerIf you want to quantify in cell culture system, provided that you have the antibody which tagged with fluorescence, then, you can do the cell staining. But you must also stain the nucleus to allow you to count the total number of cells. (His-Tag labelled cells/ Total no of cells).Usually, for this case, we use florescence microscope. It is quite tedious, but sometimes, it is better that way if the expression of you protein of interest is not high enough, which may cause difficulty to observe by Western Blot.View0 Recommendations Keya Mukherjeeasked a question related to His Tag PurificationHow purify a his-tag protein which needs a reducing agent, using a Ni column?Question30 answersMay 20, 2013I am trying to purify a His tag protein. The protein needs to have a reducing agent (1mM DTT) to be stable but it turns my column brown due to reduction of Ni. I have tried using GST fusions but somehow I do not get good protein yields with GST-columns which is why I decided to try out a Ni column. What is the best way to purify a his-tag protein that requires a reducing agent with a his-trap column?Relevant answerGary S. LacoMay 21, 2013AnswerWas trying to figure out exactly why the BME turns the column brown, found this post; I ve had this same problem and contacted GE Healthcare s tech support and it was an easy solution. The brown color comes from the reaction of the reducing agent with the free nickel that is bound to the column. Pre-wash the column with high mM imidazole elution buffer first and then equilibrate with binding buffer. If you do this, the column won t turn brown. So the BME and DTT are reacting with the un-coordinated free nickle on the column, and a 0.5 M immidazole buffer will wash it off. Just make sure to get rid of all the immidazole before loading your sample.View48 Recommendations2AdvertisementJoin ResearchGate to find the people and research you need to help your work.20+ million members135+ million publications700k+ research projectsJoin for freeSimilar topicsProtein PurificationProtein Purification TechniquesRecombinant Proteins Production PurificationHis-TagRecombinant Protein Expression and PurificationPCRWestern BlotProtein ExpressionMolecular Biological TechniquesProteinsDidn t find what you re looking for?Search for more research, methods, and experts in other areas on ResearchGate.Discover more research orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! 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