GSTFusionProteinPrep

Italicsindicateoptionalstepsespeciallyusefulfortheanalysisofuntestedproteins.

Add100ulampicillinto100mlLB.Inoculateandgrowovernight.
Inthemorning,addovernightbrothto900mlprewarmedLB.Grow1hourortoA600greaterthan0.5.Ampicillincanbeaddedtohelpmaintaintheplasmid.Makeglycerolstockfromovernightcultureifneeded!
Add1ml0.1MIPTG(final0.1mM)tothe1Lculture.Grow3to5hoursortoA600greaterthan1.0.Priortoinduction,remove100ulcellsandadd100ulSDS-PAGEsamplebuffer.Boilbrieflyandload10ulforSDS-PAGE.SharperbandscanbeobtainedifyoupassthroughsyringeseveraltimestofragmentDNA.Collecthourlysamplestodetermineoptimalinductiontime.
Pourcultureintotwo500mlcentrifugebottlesandpelletat5K10\"4C.
ResUSPendin10mlresuspensionbuffer.KeepbugsoniceasmuchaspossIBLefromhereonouttominimizeproteolysis.
LysebacteriabypassingthroughtheFrenchPress2x@12,000psi.AddPMSFifdesiredjustbeforepressing.Save100ulofpressedlysate.Pellet10K10\".Load2.5ulsupernatantforSDS-PAGE.Alsoresuspendpelletandload2.5ultocheckwhethertheproteinisinsoluble.
Transferpressedlysatetoa15mlcentrifugetube.Pelletcellulardebrisat10K10\"4C.Aliquotsupernatantandstoreat-70C.

Incubatesolublelysatewith1/2volumeof50%GSTbeadsat4CrockingontheNutatorfor30-60minutes.50mlconicalFalcontubesworkwell.
PourintoaBioRadColumnanddrainlysate.Save100ulofdepletedlysate.Load2.5ulforSDS-PAGE.
Washbeadswith3columnvolumesresuspensionbufferand1volumethrombincleavagebuffer.
Sealthebottomofthecolumn.Add1columnvolumethrombincleavagebufferplusthrombin.10ugpermgfusionproteinisagoodstartingpoint.Incubate30-60minutesatroomtemprockingontheNutator.Iftheproteinissubjecttodegradation,trya60minutecleavageat4C.
DrainBioRadcolumns.Solublecleavedfusionproteinwillbeinthefiltrate.Addacolumnvolumeofbuffertorinseoutallcleavedprotein.Rinseandsavelabelledtubesforre-use.Saveabout20ulofbeads.Load1-10ulforSDS-PAGE.
Optional:asecondthrombincleavagein1/2thepreviousvolume.
InactivatethrombinwithAEBSForPMSF.ConcentrateontheCentriconorCellStirrerifneeded,orfreezeasis.Save100ulofunconcentrated,purifiedprotein.Load1-10ulforSDS-PAGE.Quantitatebycomparisonto2.5,1.0,and0.25mgBSAstandardsonthesamegel.

Thawout1mlaliquotoffusionproteinlysate.Centrifugetoremoveprecipitate.
Incubate1mllysatewith0.4ml50%GSTbeads.Incubateat4Cwithmixingfor1hour.
Centrifuge5K2\"in1.5mlEppendorftubes.Washthebeads4XwithPBST(0.01%bMEoptional)and2Xwiththrombincleavagebuffer.Stophereifproteinboundtobeadsisneededforbindingstudy.
Resuspendthebeadsin150ulthrombincleavagebufferandadd1.4ugthrombin.Incubateatroomtemperaturewithmixingfor1hour.
Recoverthesupernatantcontainingthefusionproteinbycentrifugation.Stopthereactionwith0.7ulPMSF.Incubateatroomtemperature15\".Addanother0.7ulPMSFandincubateanother15\".

PBST:PBS(Phosphate-BufferedSaline)+0.05%Tween20
ResuspensionBuffer:PBST+2mMEDTA+0.1%bME
ThrombinCleavageBuffer:AnybufferbetweenpH7-8.5withCa++isadequate.Try50mMTRISpH8.0+150mMNaCl+2.5mMCaCl2+0.1%bME
50%GlutathioneAgaroseBeads(\"GSTBeads\")1.85gbeadsper50ml:Rehydratebeadsinlargevolumeofwaterforatleastanhour.WashwithseveralvolumesPBSTthroughWhatmanfilteronaBuchnerfunneltoremovemaltosestABIlizer.CollectgelandaddequalvolumeofPBST.Storeat4C.Donotfreeze-beadsarefragileandicecrystalswilldestroy.
Ampicillin:1.0g/10mlsterileH2O,filter0.22mM,storeat-20Cin1mlaliquots.Use1:1000.
IPTG:0.1Mstocksolution:0.238g/10mlsterileH2O,storeat-20Cin1mlaliquotsAlternate5xsolution:5g/42ml
PMSF:0.3MstocksolutioninDMSO;use1:1000
Kanamycin:0.25g/10ml,use1:1000onlyforcellswith[pREP-4]plasmid!

bMEshouldbefreshlyaddedwhencalledfor,asitisrapidlyoxidized.
PMSFshouldbeaddedjustpriortolysisofbacteria,asitisunstableinaqueoussolutions.
InsteadofPMSFtryless-toxicAEBSF.Stocksolution:0.2MinH2O,use1:100
nSec1,astickyprotein,givesahigheryieldif0.5MNaClisaddedtotheresuspensionbufferand0.05%Tween20totheThrombinCleavageBuffer.Sincetheresuspensionbufferisalready150mMNaCljustadd0.35MNaCl.Ifyouintendtofreezethepressedlysate,leaveinthecelldebris.Centrifugeitoutpriortopurification.
StirredCellConcentrator:SoaknewfilterinddH201hour.Storeat4Cin20%ethanol.

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