Description
During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies, peptides, proteins or other reactive ligands to the surface of liposomes. In general, the conjugation can be achieved through the N-terminus, the C-terminus or the available sulfur (e.g. Fab’ fraction or thiolated antibodies). Not all chemistries have the same yield and efficiency of conjugation and often reproducing biocompatible batches can be a challenge.
It is well established that electron-rich ligands such as histidine, tryptophan or cysteine show a relatively high affinity to bond with electropositive transition metals, including Co+2, Ni+2, Cu+2 and Zn+2. This observation has been exploited to improve and control the association of diverse histidine-tagged peptides to liposomes containing metal-chelating lipids. Therefore, immunoliposomes can be generated using nickel-chelating lipids such as Ni-nitrilotriacetic acid (NTA) and 1,2-dioleoyl-sn-glucero-3-[N-(amino-1-carboxypentyl)-iminodiacetic acid) succinyl] (DOGS-NTA aka DGS-NTA), which are commercially available, and His-tagged proteins or peptides. When the liposomes with Ni-NTA headgroups are combined with the His residues, typically at the N- or C-terminus of proteins, the proteins reversibly anchor to the liposomes.
Immunosome®-NTA(Ni) is a non-PEGylated product. For the other reactive (PEGylated and non-PEGyalated products) Immunosome® products suitable for other types of conjugation method see here.
Formulation Information
Immunosome®-NTA(Ni) (Non-PEGylated)
| Lipid Composition | Concentration (mg/ml) | Concentration (mM) | Molar Ratio Percentage |
|---|---|---|---|
| Total | 14.84 mg/ml | 22.45 mM | 100 |
| L-alpha-Phosphatidylcholine | 12 | 15.5 | 69 |
| Cholesterol | 2.6 | 6.73 | 30 |
1,2-Dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) | 0.24 | 0.22 | 1 |
| Buffer and Liposome Size | Specification |
|---|---|
| Buffer | Phosphate Buffered Saline |
| pH | 7.4 |
| Liposome Size | 100 nm |
Conjugation Protocol
Materials and Equipment
In order to conjugate your antibody or protein tagged with FBP to Immunosome®-NTA(Ni) liposomes you will need:
- Laboratory vortex mixer is recommended to have.
- Laboratory magnetic stirrer is needed for dialysis.
- Float-A-Lyzer® with a proper MWCO that easily allows the cleanup of your liposome conjugated ligand from free and non-conjugated protein/peptide/ligand. You need to make sure that the MWCO is below 1,000,000 dalton. At 1,000,000 dalton, the pore size on the dialysis membrane gets close to 100 nm and therefore your liposomes can be dialyzed out. You cannot use dialysis cassettes blindly. Please understand the technique before using either spin column or dialysis cassette. If you do not use the correct MWCO you can lose your entire prep. For this protocol we recommend MWCO of 300,000 dalton.
Preparation Method
- The total lipid concentration in Immunosome®-NTA(Ni) is 22.45 mM. 1% mol of the lipid in liposomes contains NTA(Ni) group and only half of them are exposed to the outside of the liposomes, which is equal to 0.11 mM of reactive conjugable lipid. For the 2 ml volume liposomes, this is equal to 2.20×10-7 mol, and for the 5 ml volume liposomes this is equal to 5.50×10-7 mol of NTA(Ni). Add 1:1 molar ratio of His6-tagged protein, peptide or antibody to NTA(Ni) lipid. Incubate Immunosome®-NTA(Ni) with His6-tagged antibody, protein, peptide or ligand for 1 hour at room temperature.
- Remove the non-conjugated antibody, protein, peptide or ligand by dialysis. We prefer dialysis to size exclusion columns. Dialysis is a much slower process but there will be minimum loss of immunoliposomes after the prep is cleaned from non-conjugated protein/peptide/ligand. Spin columns are much fast, but you can easily lose over 50% of the liposomes on the spin column. We recommend using Float-A-Lyzer® dialysis cassette from Spectrum Labs. You need to choose a cassette with proper MWCO depending on the MW of your protein, ligand, antibody or antibody fragment. NOTE: If you decide to use a dialysis cassette, you need to make sure that the MWCO is below 1,000,000 dalton. At 1,000,000 dalton, the pore size on the dialysis membrane gets close to 100 nm and therefore your liposomes can be dialyzed out. You cannot use dialysis cassettes and spin columns blindly. They come in various sizes and you need to choose the correct size wisely. Dialyze the immunoliposome solution in 1 liter of PBS at pH 7.4 for 8 hours. Change the dialysis buffer with a fresh 1 liter of PBS and let is dialyze for another 8 hours. After this step your cleaned up immunoliposome is ready to be used.re using either spin column or dialysis cassette. If you do not use the correct MWCO you can lose your entire prep.
Liposome Particle Calculator
Immunosomes are unilamellar liposomes and sized to 100 nm. The molar concentration of liposome is 22.45 mM. By having liposome diameter (nm) and lipid concentration (µM), you can calculate the total number of the lipids in one liposome and the number of the liposomes in one milliliter of the liposome solution. To use the calculator click here.
Technical Notes
- Higher concentration of DOGS-Ni-NTA lipid in the liposome formulation will cause an increased change of nonspecific binding and therefore it is important to keep the molar percentage of chelating DOGS-Ni-NTA lipid below 3% to avoid nonspecific binding. Immunosome®-Ni-NTA and Immunodox®Ni-NTA kits all contain 1% DOGS-Ni-NTA which is an optimized concentration for specific binding to His6-tagged proteins, peptides and antibody fragments.
- The binding reaction between a His6-tagged peptide and DOGS-NTA-Ni liposomes will be inhibited in the presence of high concentration of imidazole (~166 mM).
- It should be noted that the use of a 10-histidine tag promoted liposome–liposome crosslinking in the absence of PEG-modified lipids. This could be explained by suggesting that the stretch of 10 histidine residues would be sufficient to bind more than one DOGS-NTA-Ni liposome.
- It has been demonstrated that the presence of PEG polymers inhibits surface reactions between liposomes, and thus can be used to prevent aggregation.
- If you are using a ligand or peptide that is hydrophobic then it is recommended to solubilize it in DMSO or DMF and then add the buffer to it. It is recommended not to use more than 5% volume of DMSO or DMF in the solution. DMF and DMSO are both compatible with liposomes and they are also miscible in water. Other organic solvent such as ethanol and chloroform are not compatible with liposomes and will cause the liposomes to lyse. If you end up using DMSO or DMF then after the conjugation reaction is done, you need to remove DMSO and DMF from the liposomes. In order to do that you need to use a dialysis cassette that is made from REGENERATED CELLULOSE MEMBRANE. NOTE: Not all membranes are compatible with DMF and DMSO. We recommend using a Slide-A-Lyzer™ MINI Dialysis Device with MWCO of 2K made from regenerated cellulose membrane manufactured by ThermoFisher. After DMSO or DMF is removed you can use Float-A-Lyzer® dialysis device for the final step of cleaning up the prep.
- Liposomes should be kept at 4°C and NEVER be frozen.
Database
Direct link to the database page for easy navigation: Immunoliposomes Conjugation Database
Appearance
Immunosome®-NTA(Ni) is a white translucent liquid made of nano size unilamellar liposomes. Usually due to the small size of liposomes no settling will occur in the bottom of the vial. The liposomes are packaged in an amber vial.
Ordering/Shipping Information
- All liposome based formulations are shipped on blue ice at 4°C in insulated packages using overnight shipping or international express shipping.
- Liposomes should NEVER be frozen. Ice crystals that form in the lipid membrane can rupture the membrane, change the size of the liposomes and cause the encapsulated drug to leak out. Liposomes in liquid form should always be kept in the refrigerator.
- Clients who order from outside of the United States of America are responsible for their government import taxes and customs paperwork. Encapsula NanoSciences is NOT responsible for importation fees to countries outside of the United States of America.
- We strongly encourage the clients in Japan, Korea, Taiwan and China to order via a distributor. Tough customs clearance regulations in these countries will cause delay in custom clearance of these perishable formulations if ordered directly through us. Distributors can easily clear the packages from customs. To see the list of the distributors click here.
- Clients ordering from universities and research institutes in Australia should keep in mind that the liposome formulations are made from synthetic material and the formulations do not require a “permit to import quarantine material”. Liposomes are NOT biological products.
- If you would like your institute’s FedEx or DHL account to be charged for shipping, then please provide the account number at the time of ordering.
- Encapsula NanoSciences has no control over delays due to inclement weather or customs clearance delays. You will receive a FedEx or DHL tracking number once your order is confirmed. Contact FedEx or DHL in advance and make sure that the paperwork for customs is done on time. All subsequent shipping inquiries should be directed to Federal Express or DHL.
Storage and Shelf Life
Storage
Immunosome® products should always be stored at in the dark at 4°C, except when brought to room temperature for brief periods prior to animal dosing. DO NOT FREEZE. If the suspension is frozen, the encapsulated drug can be released from the liposomes thus limiting its effectiveness. In addition, the size of the liposomes will also change upon freezing and thawing.
Shelf Life
Immunosome®-NTA(Ni) is made on daily basis. The batch that is shipped is manufactured on the same day. It is advised to use the products within 4 months of the manufacturing date.
References and background reading
1. Faham, Abdus, and Joseph Altin. “Ag‐bearing liposomes engrafted with peptides that interact with CD11c/CD18 induce potent Ag‐specific and antitumor immunity.” International journal of cancer 129, no. 6 (2011): 1391-1403.
2. Faham, Abdus, David Bennett, and Joseph G. Altin. “Liposomal Ag engrafted with peptides of sequence derived from HMGB1 induce potent Ag-specific and anti-tumour immunity.” Vaccine 27, no. 42 (2009): 5846-5854.
3. Bak, Martin, Rasmus I. Jølck, Rasmus Eliasen, and Thomas L. Andresen. “Affinity Induced Surface Functionalization of Liposomes Using Cu-Free Click Chemistry.” Bioconjugate chemistry 27, no. 7 (2016): 1673-1680.
4. Chikh, Ghania G., Wai Ming Li, Marie-Paule Schutze-Redelmeier, Jean-Claude Meunier, and Marcel B. Bally. “Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids.” Biochimica Et Biophysica Acta (BBA)-Biomembranes 1567 (2002): 204-212.
5. Rüger, Ronny, Dafne Müller, Alfred Fahr, and Roland E. Kontermann. “Generation of immunoliposomes using recombinant single-chain Fv fragments bound to Ni-NTA-liposomes.” Journal of drug targeting 13, no. 7 (2005): 399-406.
6. Platt, Virginia, Zhaohua Huang, Limin Cao, Matthew Tiffany, Kareen Riviere, and Francis C. Szoka Jr. “Influence of multivalent nitrilotriacetic acid lipid− ligand affinity on the circulation half-life in mice of a liposome-attached his6-protein.” Bioconjugate chemistry 21, no. 5 (2010): 892-902.
7. Christis, Chantal, and Sean Munro. “The small G protein Arl1 directs the trans-Golgi–specific targeting of the Arf1 exchange factors BIG1 and BIG2.” J Cell Biol (2012): jcb-201107115.
8. Shao, Shuai, Jumin Geng, Hyun Ah Yi, Shobhit Gogia, Sriram Neelamegham, Amy Jacobs, and Jonathan F. Lovell. “Functionalization of cobalt porphyrin–phospholipid bilayers with his-tagged ligands and antigens.” Nature chemistry 7, no. 5 (2015): 438-446.
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
原理简介：
本试剂盒采用改进SDS-碱裂解法裂解细胞，离心吸附柱内的硅基质膜在高盐，低pH值状态下选择性地结合溶液中的质粒DNA，再通过去蛋白液和漂洗液将杂质和其它细菌成分去除，最后低盐，高pH值的洗脱缓冲液将纯净质粒DNA从硅基质膜上洗脱。
注意事项：
◆ 第一次使用时，将试剂盒所带全部的RNase A加入溶液P1后（终浓度100ug/ml）置于4℃保存。如果溶液P1中RNase A失活，提取的质粒可能会有混杂有微量RNA残留， 这时可在溶液P1中补加RNase A即可。
◆ 第一次使用前请先在15ml漂洗液WB中加入45ml无水乙醇，加入后请及时在方框打钩标记已加入乙醇，以免多次加入!
◆ 温度低时溶液P2中SDS可能会出现浑浊或者析出沉淀，可在37℃水浴加热几分钟，即可恢复澄清，不要剧烈摇晃，以免形成过量的泡沫。
◆ 避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化，各溶液使用后应及时盖紧盖子。
试剂盒特点：
◆ 产量高---一次提取高达30ug以上的质粒。
◆ 纯度高---OD260/OD280一般为1.80~1.85本试剂盒提取的质粒纯度好，能充分保证测序所需要的读长(用于ABI3730测序一般可达1000bp有效读长)。
◆ 快速，方便，不需要使用有毒的苯酚，氯仿等试剂，也不需要乙醇沉淀。
提示
BIOTEKE的质粒提取试剂盒既适用于革兰氏阴性菌中质粒的提取，同时也可从革兰氏阳性菌中提取质粒。由于革兰氏阳性菌外被一层较厚的细胞壁，会严重阻碍细菌细胞的裂解，因此必须在裂解细胞前破除，方法如下：
收集适量的菌体，加入250ul溶液P2，充分悬浮菌液，加入溶菌酶使其终浓度在10-20mg/ml左右在37℃处理30分钟左右。加入溶菌酶的浓度和处理的时间可根据不同的菌主和具体实验条件进行调整。
转膜后就要区分与胶接触的一面与另一面了。话说楼主好奇的话可以跑蛋白的试试看哈。比如目的蛋白的胶用光滑面贴着胶，内参蛋白用粗糙面贴着胶一起转膜试试转膜结果看看呢。
1、如果是提取的总蛋白，然后做WB，用β-actin或者GAPDH做内参肯定是没有问题的，这是公认的东西。
2、如果用膜蛋白提取试剂盒提取蛋白，再用β-actin作为内参似乎不妥，因为理论上来讲β-actin在膜上是不表达的。WB能做出β-actin来是因为膜蛋白提取时把胞质蛋白也提出来了。然而，如果我们试验目的是用药物处理细胞，比较处理前后某种膜蛋白的表达情况，此时用膜蛋白提取试剂盒提取膜蛋白后再用β-actin做内参似乎就不妥了，因为你根本不知道药物处理前后混杂了多少的胞质蛋白进来。如果没有胞质蛋白混进去的话，β-actin就是检测不到的。
