1、020多肽溶解方法和步驟轉載標簽：分類：多肽知識多肽溶解方法吉爾生化沒有絕對理想的一種溶劑可以做到既能溶解所有多肽,又能保持它們的完整性并且與生物學檢測相一致.因此,不得不嘗試一系列強溶劑直到多肽溶解.對有溶解困難的多肽,下列方法也許有所幫助咨詢Option21PEPscreenPeptideSetsOption11t.AfdJDOjiLoFK%HOAc-vat?FJSencarte七：5t詔And2DQliLofDMSCcrDMFSc-nicatefor5mirutes02002090%Dissolved】，AdduLcfACNts.iju.jJ：?certdes2.Scricatefc-r

2、5minLtes1_化”：zasLclecastdespectJei?fDi55cl7eir1DDDMSO1Scnicatefcr5minutesRiptideSolutionbjcrma1?evcLmecccnc?-ra：crvtil啟喊!rujbuffeHRsptideStockSolution4PeptideStorage4第一步總的來說,先用無菌水或者稀醋酸（0.將1多%肽）溶解到一個較高濃度下作為儲存溶液,而不是直接配置到檢測濃度等需要用的時候再把這個儲備溶液用緩沖配制到需要濃度比如一條多肽要溶解成的中我們需要先將多肽溶解成2的儲備溶液然后在使用前先移取00的0再加入00的無菌水最后

3、加入00的2儲備液千萬不要直接用緩沖液來溶解多肽因為很多肽在高鹽濃度下溶解度是下降的,而且如果發生不溶情況,去除這些鹽和有機試劑是很困難的一件事.如果多肽溶解過程中出現可見顆粒始終無法分散到水相中,可以用超聲的辦法來破碎.不過超聲只能加速溶解,并不能起到改善溶解性的效果第二步溶解前先審視一下多肽序列,如果多肽中下列氨基酸占比比較高這條多肽基本上是難溶的另外要注意計算一下序列中含有多少正價基團和端和負價基團和端在中性條件下最后的凈價是正是負價態為正的在溶解時可以用稀醋酸調節到酸性價態為負的在溶解時可以用稀氨水調節到堿性如果這樣仍不能溶解可以考慮凍干去除溶劑后變成用強有機溶劑來溶解第三步如果你的序

4、列在任何下都基本不帶電荷或者說你的序列中疏水性氨基酸含量超過0甚至更高前面兩步基本上是多余的直接考慮加入少量乙腈乙醇或者來溶解甚至可以同鹽酸胍和脲來分散多肽.這些方法溶解多肽的濃度取決于你最終生物檢測需要.如果已經知道一條多肽在水相中溶解不是太好而最終使用卻必須在水相可以考慮全部用醋酸或者來溶解然后緩慢加水來稀釋這樣有機溶劑有助于多肽分散到水相中多肽溶液的儲存問題溶解的多肽的保質期是比較有限的特別是含有和的多肽為了延長保存時間使用無菌水保持適當酸性還有分裝冷凍在20r或者更低是建議采用的方法。另外，一定要避免反復凍融，這個對多肽的損傷最大。0,thissolutionslowlywithwat

5、ertogiveapproximately10%(v/v)DMF.Ifthepeptideprecipitatesatanystageduringthisstep,stopaddingwaterandaddalittlemoreDMFuntilthepeptideredissolves.Suchpeptidesmaybetooinsolubleinwatertobeusedatconcentrationsequaltotheothersintheset.5.Diluteeachsolubilizedpeptidewiththesolventfoundtobeeffectiveforit,tob

6、ringthestocksolutionstothesamepeptideconcentration.Thissimplifiescalculationsandsubsequenthandling.Furtherdilutions,asneededforthebioassay,canbemadeintheassaybuffere.g.asadilutionseries(titration).Dilutionofarelativelyinsolublepeptidewithbufferatthisstepmaysuccessfullyavoidprecipitationbecauseitisno

7、watalowconcentration(belowitssolubilitylimit).6.ExceptafteradditionofDMF,allsolutionscanbeeasilylyophilisedtoreturnthepeptidetoaformsuitableforlongtermstorage,ifrequired.Thisisonlyoneofalargenumberofpossibleprocedures.Theonechosendependsontheassaysystem,andtheneedforaparticularbufferorpeptideconcent

8、ration.ContactMimotopesforfreetechnicaladviceifyouwishtouseaparticularbuffernotmentionedhere.AStrategyforDissolvingLargeSetsofPeptidesPeptidesetssuchasaGeneralnet(Gnet),Replacementnet(Rnet)etc.formassscreeningaresuppliedaslyophilisedsolidsinpolypropylenetubes.Inthesecases,theonlypracticalmethodmaybe

9、toapplyonesolventtoallpeptidesandusethesolutionsobtainedwithouttryingtooptimizeforeachpeptide.Forexample,forTcelldeterminantmapping,wehavefoundthatasolventcomprising0.1MHEPESbufferpH7.4ina40%acetonitrile/watersolutiongiveseffectivesolutionsofmostpeptidesandisnontoxicwhendiluted20-foldormoreinanassay

10、.Inthiscase,sonicationofthetubesisalsoadvisabletomaximisethedissolutionofthepeptides.Ifacceptableintheassaysystemattheintendedworkingconcentration,agoodgeneralsolventsuchasDMForDMSOcouldalsobeusedasthefirstreagentaddedtoallpeptidesintheset.Onspecialrequest,peptidesetscanbeshippedintheformoffrozensol

11、utions,avoidingtheproblemsofredissolvingthepeptides.Shippingfrozenmaterialsaddssubstantiallytothecostofthepeptidesandcancreateproblemsifthepeptidesaredelayedintransit.ChemicalChangesinYourPeptidesPeptidesvaryinstability,andapeptideassuppliedmaysoonbedegradedifcareisnottakentoensureproperstorage.Inad

12、ditiontotheriskofdegradationfromproteolyticenzymes,otherchemicalchangescanoccur.Theshortsectionbelowismeanttohelpwithsituationswhichwillcommonlyarise.1.OxidationAcharacteristicofcysteine-andmethionine-containingpeptidesisthetendencyoftheseresiduestooxidise.Susceptibilitytooxidationissequence-depende

13、ntandsometimesevenminimalexposuretoairofpeptidescontainingtheseaminoacidscanleadtooxidation.Ifyouwishtoavoidoxidation,alwaysworkwithdegassedordeoxygenatedsolventsandsolutions.Ifpossible,maintainpeptidesolutionsatacidicpH(7).RateofoxidationincreaseswithpH,soevenifthepeptideisinthefullyreducedforminit

14、ially,someoxidationwilloccurifthepeptideismaintainedunderneutralorbasicconditions.Normally,singlepeptidesareconsignedinthefullyreducedform.Ifhandledproperly,theywillremainso,butifyouneedtocarryoutreductionofapeptidetheproceduresareasfollows:1.Reductionofoxidisedcysteine.Dissolvethepeptidein0.1Mammon

15、iumbicarbonatecontainingdithiothreitol(approximately10-50foldmolarexcess)andholdfor4hatroomtemperature.Thisprocedureisareasonablestartingpointbutcertainsequencesmayrequiremoreforcingconditionsoftemperatureandtime.Afterreduction,lyophilisethesolution,orde-saltusingsizeexclusiongelchromatography(e.g.P

16、harmaciaSephadexG-25)orreverse-phasechromatography(e.g.Millipore/WatersSep-Pak).Topreventre-oxidation,followthehandlingproceduresmentionedaboveandstorethepeptidepowderundernitrogengasandinafreezer.2.ReductionofoxidisedmethioninebythemethodofHoughtenandLi2.Dissolvethepeptidein10%aceticacidinwater,and

17、addN-methylmercaptoacetamideto10%(v/v).Incubateat37degreesCfor24hormore,thenlyophiliseorde-saltasforcysteine-containingpeptides.2.OtherreactionsPeptideshaveavarietyofreactivesidechains,andsidereactionscanoccurunderbothacidicandbasicconditions.Forexample,ifglutamineorglutamicacidoccursattheN-terminus

18、ofapeptide,cyclisationtoformpyroglutamateislikelyunderacidicconditions(10%aceticacid).Mildbasicsolutions(0.1Mammoniumbicarbonate)willleadtoimideformationinasparagine-containingpeptides.Whendissolvingsinglepeptides,avoidconditionsknowntopromotesidereactionswiththeresiduespresent.Degradationduetomicrobialgrowthshouldnotoccurprovidedsteriledistilledwaterorbuffers