James Hardwick CNBr Cleavage Procedure相关交流-蚂蚁淘在线

James Hardwick CNBr Cleavage Procedure

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1.Immunoprecipitatetheproteinandrunitonapreparativegel.CNBrcleavagemustbedonewithproteintransferredtoanitrocellulosefilter.NeitherImmobilonnorNyloncansubstitute.

IMPORTANT:WashtheNC2Xfor15minindeionizedH2Oafterthetransferiscomplete.ThisremovesanyresidualTris-glycinethatseemstoaffectthemigrationoftheCNBrfragmentsduringSDS-PAGE.

2.CutoutthepieceofNCcontainingtheproteinandimmediatelyputitintoamicrofugetubecontaining200microlitersof100mg/mlCNBrin70%formicacid.CNBrstocksare200milligrams/mlin70%formicacidandarestoredat-70°C.70%formicacidismadefrom98-100%formicacidandMQH2O.

Optional:Ifyouwanttoknowhowmanytotalcountsyouhaveineachmembranepiece,placethecutoutpiecesoffilterinH2Oandcountthem(Cerenkov)beforeyouplacethemintheCNBr.

3.IncubatetheproteininCNBratroomtemp.for1.5hr.Increaseddigestontimedoesnotincreaseyield.4.Spin1-5mininthemicrofuge.Transfersupe.toanewmicrofugetube.5.SpeedVacdry(30minfor200microliters).6.ResUSPendin500microlitersofMQH2Oanddryonthespeedvacagain(approx2hrs.).Thisshouldgetridoftheresidualformicacid.

Kunxinresuspendedin40microlitersinordertosavetime.Resuspendinginlargervolumesremovesmoreoftheformicacidandresultsinbetterresolutionofthecleavageproducts.

7.Count(Cerenkov)boththedriedsamplesandthemembranepiecestheywerereleasedfrom.Youryieldshouldbebetween85-95%.7.Resuspendin20-40µlSDSgelsamplebuffer.Thesamplebuffershouldremainblue.Ifitturnsyellow,residualformicacidispresentandwillcauseyoursamplestorunbadly.Trisbuffer(1µlof1MTrispH8.0)canbeaddedtoraisethepH.8.Runona24%lowbisTricinegel.UserainbowMarkersasMWindicators.Ittakesapproximately9hourstorunthebromphenolblueoffofalonggelusingaconstantcurrentof50mA.Ifyouneedtoresolvemultiplebandsbelow4kD,thenuseaseparatinggelinadditiontothestackerandresolvinggel.Seethereferencebelow.9.Forreference,asamplewith100cpmmustbeexposedfor2-3dayswithascreentoseeastrongsignal.

GelRecipes:

TrisAnode(lower)Buffer1liter

0.2MTrispH8.9	24.2gTrisbase
	Bringto1literwithwater--adjusttopH8.9withHCl

TricineCathode(upper)Buffer1liter

100mMTris	12.1gTrisbase
100mMTricine	17.9gTricine
0.1%SDS	1gSDS
	MQH2Oto1000ml(don"tneedtoadjustpH)

24%lowbisgel--25ml(onelonggel)

24%Acrylamide	15ml40%Acrylamide
0.054%Bis	1.36ml1%Bis
1MTris	8.3ml3MTrisHClpH8.45
0.1%SDS	0.25ml10%SDS
Temed	20microliters100%Temed
Ammoniumpersulfate	0.20ml10%Ammoniumpersulfate

StackingGel--10ml

4%Acrylamide	1.33ml30%Acrylamide
0.1%Bis	1ml1%Bis
1MTris-HCl	3.33ml3MTris-HClpH8.45
0.075%SDS	75microliters10%SDS

PlustheusualamountsofTemedandPersulfate.

References:

Buffers,pH,stackinggelrecipefrom:

Schagger,H.andvonJagow,G.(1987).Anal.Biochem.166,368-379.

Acrylamide:BisratioforresolvinggelfromKunxin"soldprotocol.

Luo,K.,andSefton,B.M.(1990)Transferofproteinstomembranesfacilitatesbothcyanogenbromidecleavageandtwodimensionalproteolyticmapping.Oncogene5:921-923.

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