LyseGrampositiveorGramnegativebacteriaforproteinornucleicacidpurificationsfrombacteria
- HighlyActive:Thisenzymeis200Xmoreactivethaneggwhitelysozyme,thusimprovingbacteriallysisandusinglessenzyme
- Easy:Nosampleagitationorheatgenerationnecessaryduringlysiswhichhelpspreserveproteinfunction
- FlexIBLe:Usuagevolumesareeasilyadjustedbasedonthescaleoftheexperiment
Applications
- LysisofGram-negativeorGram-positivebacteria(Table1)forproteinpurification(Fig.1).
- Purificationofnucleicacidsfrombacteria.
Ready-Lyse™LysozymeSolutionisarecombinantlysozymepreparation,derivedfromanonmammalian,nonaviansource,thatisusefulforthelysisofGram-negative(suchasE.coli)andGram-positive(suchasBacillussp.)bacteria.ThespecificactivityofReady-LyseLysozymeis200-foldhigherthanthespecificactivityofegg-whitelysozymeand,therefore,lesslysozymeisneededinareaction.Also,unlikeegg-whitelysozyme,Ready-LyseLysozymeSolutionisstableat-20°C,eliminatingtheneedtoprepareafreshsolutionforeachuse.TheuseofReady-LyseLysozymeresultsinhigheryieldsofproteinthancanbeobtainedwithstandardeggwhitelysozyme.
UnitDefinition:OneunitofReady-LyseLysozymeproducesadecreaseinA350of0.001perminuteat23°Cwitha0.5mg/mlsUSPensionoflyophilizedE.coliK802cellsin50mMTris-HCl(pH7.5).
StorageBuffer:50%glycerolcontaining50mMTris-HCl(pH7.5),0.1MNaCl,0.1mMEDTA,1mMDTT,and0.1%Triton®X-100.
Table1.BacterialysedwithReady-Lyse™LysozymeSolution. |
Figure1.LysiswithReady-Lyse™Lysozymeincreasesyieldsofnucleicacids.500µgpermlofpHC79cosmidDNAwasincubatedfor15minutesat22°CinTEbuffer(25mMTris(pH8.0),10mMEDTA)containingeither5µg(30KU)permlofReady-LyseLysozymeor500µg(25KU)permlofeggwhitelysozyme.Thesolutionswerecentrifugedfor10minutesandthepelletswereresuspendedinTEbuffercontaining0.1%SDS.DNAinsupernatantsandpelletswasseparatedbyelectrophoresisina0.8%agarosegel.Approximately50%oftheDNAwaslostduetoprecipitationbyeggwhitelysozyme(EW),whileReady-LyseLysozyme(RL)causedminimalprecipitationlossesofDNAcomparedtocontrol(C)sampleswithoutlysozyme. | Figure2.UseofReady-Lyse™LysozymeSolutiontorecoverrecombinantproteins.OnemlofinducedcellsfromarecombinantE.coliclonewaspelletedbymicrocentrifugationbeforeinductionandat1and3hoursafterinduction.Eachsamplewasresuspendedin50µlofcoldTEBGbuffer.OneµlofReady-LyseSolutionwasaddedtoeachsuspensionandthecellswereincubatedatroomtemperaturefor30minutes.Thecelldebriswaspelletedand10µlofthesupernatantwererunonanSDS-PAGEgel.Lane1,molecularweightMarkers;Lanes2-4,timepointsofinduction.Theinducedproteinisdesignatedbyanarrow. |
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根据组成不同,可分为两种,弱酸及其对应的强碱弱酸盐,弱碱及其对应的强酸弱碱盐。
因为HF可以和NaOH反应生成NaF和水,当NaOH反应完之后,NaF就可以与HF组成缓冲溶液,所以说可以直接使用NaOH和HF来配制缓冲溶液。
这也是一般配制缓冲溶液的方法,也就是用强碱和弱酸(或者强酸和弱碱)来配制缓冲溶液。
(2)酸和盐浓度等比例也增减时,溶液的pH值不便。
(3)酸和盐浓度相等时,缓冲液的缓冲效率为最高,比例相差越大,缓冲效率越低,一般地说缓冲液有效缓冲范围为PK±1pH
浙江大学奚振宇、徐又一、朱利平等2009年在journal of membrane science(中文翻译为《膜科学杂志》)发表的一篇论文,其中探讨了缓冲溶液的pH值对聚乙烯-多巴胺复合膜亲水性的影响,发现缓冲溶液的pH值为8.5时制备的复合膜亲水性最强。
通过大量的实验,研究人员发现,pH值为8.5时制备的多巴胺复合膜性能最优。这个是通过大量的实验总结得到的结论。
2.尿素:有膜蛋白的损失
3.RIPA:用于磷酸化蛋白的温和裂解,不破坏磷酸基团