整理GST融合蛋白纯化方法文档格式.docx
- 文档编号:22714517
- 上传时间:2023-02-05
- 格式:DOCX
- 页数:19
- 大小:27.06KB
整理GST融合蛋白纯化方法文档格式.docx
《整理GST融合蛋白纯化方法文档格式.docx》由会员分享,可在线阅读,更多相关《整理GST融合蛋白纯化方法文档格式.docx(19页珍藏版)》请在冰豆网上搜索。
2.Lysozymesolution
10mg/mlinwater(makefresh)
3.PBS
4.ElutionBuffer
50mMTris.Cl,pH9.0
20mMGSH
5.10%SarkosylinSTEBuffer
6.10%TritonX-100inSTEBuffer
7.1MDTT
8.100mMIPTG
Procedure
Day1
1.Setupanovernightculturein50ml2XTYwith150mg/mlofampicillin.
Day2
1.Seed5mlofovernightcultureto500ml2XTYwith150mg/mlofampicillin.
2.Growat37oCtoanA600of0.6to0.8.
3.Inducewith0.1mMto2mMofIPTG.Growfor3hrat37oCorgrowovernightatroomtemperature.
LowerIPTGconcentrationsandlowergrowingtemperaturestendtoproducegreatersolubilityattheexpenseofyield.
4.Pelletcellsbycentrifugingat3000g,4oCfor10min.Decantmediaandresuspendcellsin30mlice-coldPBStowash.Transfertoa40-mlOakRidgetubeandcentrifugeat3000g,4oCfor10min.DecantPBS.
5.Thisisaconvenientpointtostopandtostorepelletsat-80oC.Elsecontinuetolysecells.
6.Thawpelletoniceifcellsarefrozenelseproceedtothenextstep.
7.Resuspendpelletin10mloficecoldSTEBuffer.
8.Add100mloffreshlypreparedlyozymesolution,incubateonicefor15min.Justbeforesonication,add100mlof1MDTTand1.4mlof10%Sarkosyl.Mixthoroughlybyinversionandsonicateforatotaltimeof1min.
9.Centrifuge16,000rpmfor20minontheSS34rotortopelletdebris.Transfersupernatanttoa50-mlconicaltubeanddiscardthepellet.Add4mlof10%TritonX-100andtopupwithSTEBufferto20ml.TheeffectiveconcentrationofSarkosylandTritonX-100willbe0.7%and2%respectively.Incubateatroomtemperaturefor30min.
10.Pourthelysateto1mlbedofpreparedGlutathioneSepharoseinPBS.Incubateatroomtemperaturefor30minto1hrwithagitation.
Topreparethe50%slurry,shakeupthemediaandpipette2mltoa50mltube.Fillto50mlwithPBS,inverttubeafewtimes.Centrifugeto2000rpmonaswingbucketcentrifugethenswitchoff.CarefullysuckoffPBSandresuspendbeadswith1mlofPBS.
11.Washthebeadswith3X50mlofPBS.Finallyresuspendin5mlofPBS.Pourtoadispo-column.Washthe50-mlconicaltubewithanadditional5mlofPBS.Poolwiththefirst5mlinthedispo-column.
Towash,usethesamecentrifugationtechniqueforpreparingthebeads.Whentransferringbeadstocolumn,donotpipettebutpour.Thebeadstendtosticktopipettetips.
12.Ifdesired,elutewith10x1mlfractionsofElutionBuffer.DeterminedesiredfractionswithSDSPAGE
亲和层析实验技术方法
INTRODUCTION
Thisprotocoldescribesamethodforremovingantibodiesthatreactwithbacteriallyencodedproteinsbypassingacrudepreparationofimmunoglobulinsthroughacolumncontainingimmobilizedbacterialproteins.
MATERIALS
∙Reagents
E.colistrainusedashostforpreparationofexpressionlibrary
Antibodypreparationthatistobeusedforscreening
ThisprotocolworksbestwhenusinganIgGfraction,preparedbychromatographyoftheantiserumonproteinA-Sepharose.
∙Celllysisbuffer
0.1Msodiumborate(pH8.0)
1MNaCl
Sterilizethecelllysisbufferusinga0.45-µ
mfilter,andstoreatroomtemperature.Approximately100mlofcelllysisbufferisrequiredper1literofbacterialculture.
∙Growthmedium
OneliterofgrowthmediumappropriatefortheE.colistrainofchoiceisrequired.
∙Lysozyme
Dissolvesolidlysozymeataconcentrationof10mg/mlin10mMTris-Cl(pH8.0)immediatelybeforeuse.MakesurethatthepHoftheTrissolutionis8.0beforedissolvingtheprotein.LysozymewillnotworkefficientlyifthepHofthesolutionislessthan8.0.
Useamolecularbiologygradeoflysozyme.Addsolidlysozymetoassistlysisofbacterialcells.
∙NaOH(1N)
Thepreparationof10NNaOHinvolvesahighlyexothermicreaction,whichcancausebreakageofglasscontainers.Preparethissolutionwithextremecareinplasticbeakers.To800mlofH2O,slowlyadd400gofNaOHpellets,stirringcontinuously.Asanaddedprecaution,placethebeakeronice.Whenthepelletshavedissolvedcompletely,adjustthevolumeto1literwithH2O.Storethesolutioninaplasticcontaineratroomtemperature.Sterilizationisnotnecessary.
∙PancreaticDNaseI
o1mg/mlPancreaticDNaseI
o50mMNaCl
o10mMTris-Cl(pH7.5)
o1mMMgCl2
Dissolve2mgofcrudepancreaticDNaseI(Sigmaorequivalent)in1mlof50mMNaCl,Tris-Cl(pH7.5),1mMMgCl2.WhentheDNaseIisdissolved,add1mlofglyceroltothesolutionandmixbygentlyinvertingtheclosedtubeseveraltimes.Takecaretoavoidcreatingbubblesandfoam.Storethesolutioninaliquotsof-20°
°
C.AddsolidDNaseItothebacterialcelllysatetodigestchromosomalDNA.
Tris-bufferedSaline(TBS)
∙Dissolve8gofNaCl,0.2gofKCl,and3gofTrisbasein800mlofdistilledH2O.Add0.015gofphenolredandadjustthepHto7.4withHCl.AdddistilledH2Oto1liter.Dispensethesolutionintoaliquotsandsterilizethembyautoclavingfor20minutesat15psi(1.05kg/cm2)onliquidcycle.Storethebufferatroomtemperature.
∙TBScontaining0.2%(w/v)sodiumazide
∙TritonX-100
METHOD
Growa1-litercultureoftheappropriatestrainofE.coli(e.g.,Y1090hsdR,XL1-Blue,orDH1)tostationaryphase.
∙Recoverthebacteriabycentrifugationat4000g(5000rpminaSorvallGSArotor)for20minutesat4°
C.
∙Pouroffthemedium,andstandthecentrifugetubesinaninvertedpositiontoallowthelasttracesofmediumtodrainaway.
∙Resuspendthepelletin100mlofCelllysisbuffer.
∙Add200mgoflysozyme,andincubatethebacterialsuspensionfor20minutesatroomtemperature.
∙Add1mgofpancreaticDNaseIand200µ
lofTritonX-100.
∙Incubatethebacterialsuspensionfor1hourat4°
C,oruntiltheturbidityclearsandtheviscositydecreases.
∙Centrifugethebacteriallysateat8000g(8200rpminaSorvallSS-34rotor)for20minutesat4°
C.Carefullydecantthesupernatantintoafreshflask.
∙AdjustthepHofthesupernatantto9.0with1MNaOH.
∙DeterminetheconcentrationofproteininthelysateusingtheLowry,Bradford,orothermethodofmeasurement.
∙Chilltheextractto0°
C,andthenbindthebacterialproteinstocyanogen-bromide-activatedSepharose4BortoAffi-Gel10accordingtothemanufacturer'
sinstructions.
∙Beforeuse,equilibratetheSepharose4BorAffi-Gel10resincontainingconjugatedE.coliproteinsinTBScontaining0.2%(w/v)sodiumazide.
∙Use1mlofsettledvolumeofresincoupledtoE.coliantigenforeachmilligramofIgGproteintobepurifiedbyaffinitychromatography.MixtheIgGandthecoupledresinandincubatefor12-18hoursatroomtemperatureonarotatingwheeldevice.
Loadtheslurryintoachromatographycolumn.RecovertheantibodybywashingthecolumnwithTBS.Collectfractions(0.2columnvolumeeach)untiltheOD280dropstozero.Poolthefractionscontainingantibody,andstorethepoolat-20°
Cuntilitisusedforimmunologicalscreening.
REFERENCES
1.deWet,J.R.,Fukushima,H.,Dewji,N.N.,Wilcox,E.,O'
Brien,J.S.,andHelinski,D.R.1984.Chromogenicimmunodetectionofhumanserumalbuminand{alpha}-L-fucosidaseclonesinahumanhepatomacDNAexpressionlibrary.DNA3:
437-447.[Medline]
Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliabilityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:
MolecularCloning:
ALaboratoryManual,ThirdEdition,JosephSambrookandDavidW.Russell,©
2001byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.14.28-14.30.
GST融合蛋白纯化方法
1
目的片段接入pGEX载体;
2涂板,挑单克隆,摇菌至OD600≈1.0,加入IPTG(终浓度1mM)诱导6-8h;
3收菌,每升菌液约以50mLPBS重悬,加入1%TritonX-100(v/v),1%β-巯基乙醇(v/v),PMSF(终浓度1mM);
以下步骤均在冰上操作:
4超声破碎菌体,15000g,10min离心取上清,在上清中加入适量GST-beads,轻轻晃动令其吸附蛋白1h;
52000g,3min离心弃上清;
6加入至少10倍体积PBS,轻摇至beads悬浮于溶液中,2000g,3min离心弃上清;
7重复步骤6两次;
8加入1mLGSTElutionBuffer,轻摇10min;
92000g,3min离心,收集上清;
10重复步骤8-9至少两次;
11SDS-PAGE电泳检测蛋白纯度,Bradford法检测蛋白浓度;
12将蛋白置于-20℃保存。
P.S.大量提取前应取少量菌液,改变IPTG浓度,诱导温度,诱导时间等,以确定蛋白表达的最适条件。
GSTfusionproteinpurificationGST融合蛋白纯化
1)grow20mlcellsO/N37℃dilute50XintoprewarmedLB,growto0.6ODorabout1hr.Inducew/2mMIPTG(238mg/0.5l),grow3hr,spin6kGS310'
freezeat70℃
2)extractcells(from500ml)in25mls.HeintzBufferplustriton(HBT)bygentlepipetteresuspendingonicecirca10'
afterthawing.
3)transferto50mlconicalss34fliptoptubes.
4)add10mglysozymepowdertothe50ml(cellsfrom1liternowin150mltube),digest15'
onice.
5)sonicatewithlargeprobe1'
80%power,freezeinlN,thawat37℃sonicate
againonice,solutionshouldbecomeviscous.
6)add1mgDNAseandRNAse,incubateonice15'
.
7)spin7.5krpm4℃ss3410'
8)transfersupernatantstoconicalscrewcaps,freezeinlN2,maystore.
9)Batchadsorbw/4ml50%slurryGT-Sepharose(PL17-0756-01),1hr,4℃spin2'
4konbench.
10)aspirate,resuspendin25mlHBT,spin,repeat.
11)pourslurryintocolumn(Econo1.7x20),elutetotop,thenwith20columnvols.ofHB-T.
12)eluteproteininminimalvolume(
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- 整理 GST 融合 蛋白 纯化 方法