染色质免疫共沉淀 ChIP Protocol及crosslink 的原理.docx
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染色质免疫共沉淀 ChIP Protocol及crosslink 的原理.docx
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染色质免疫共沉淀ChIPProtocol及crosslink的原理
ChIPProtocol
I.Formaldehydecross-linkingofcells
Use5x107–1x108cells(70-80%confluencyforadhesioncellsof8-1215cm2platesor175cm2flasks)foreachimmunoprecipitation.
Adherentcells:
1.Add1/10volumeoffresh11%FormaldehydeSolutiontoplates.
2.Swirlplatesbrieflyandletthemsitatroomtemperaturefor10minutes.
3.Add1/20volumeof2.5Mglycinetoplatestoquenchformaldehyde.
4.Rinsecellstwicewith5ml1xPBS.Harvestcellsusingsiliconscraper.
5.Poolcellsin50mlconicaltubesandspinat1,350xgfor5minutesat4°C(SorvallLegendRTcentrifugewithswingingbucketrotor).Discardsupernatantandresuspendpelletin10ml1xPBSper108cells.
6.Transfer5x107–1x108cellsto15mlconicaltubeandspinat1,350xgfor5minutesat4°C(SorvallLegendRTcentrifugewithswingingbucketrotor).Discardsupernatant.
7.Flashfreezecellsinliquidnitrogenandstorepelletsat–80°C.
Suspensioncells:
1.Add1/10volumeoffresh11%FormaldehydeSolutiontoflasks.
2.Swirlflasksbrieflyandletthemsitatroomtemperaturefor20minutes.
3.Add1/20volumeof2.5Mglycinetoflaskstoquenchformaldehyde.
4.Spincellsat1,350xgfor5minutesat4°C.
5.Poolcellsinrequirednumberof50mlconicaltubesandspinat1,350xgfor5minutesat4°C(SorvallLegendRTcentrifugewithswingingbucketrotor).Dumpsupernatant.
6.Resuspendcellsin50ml1XPBS,spinat1,350xgfor5minutesat4°C.Discardsupernatant.Repeatonce.
7.Resuspendin10mlper108cells.Aliquot5x107–1x108cellsto15mlconicaltubesandspin1,350xgfor5minutesat4°C(SorvallLegendRTcentrifugewithswingingbucketrotor).Discardsupernatant.
8.Flashfreezecellsinliquidnitrogenandstorepelletsat–80°C.
FormaldehydeSolution
FinalConc.StockFor50ml
50mM1MHepes-KOH,pH7.52.5ml
100mM5MNaCl1.0ml
1mM0.5MEDTA50.0µl
0.5mM0.5MEGTA100.0µl
11%37%Formaldehyde14.9ml
ddH2O31.5ml
II.Preblockandbindingofantibodytomagneticbeads
NOTE:
ExacttypeofDynalbead(ProteinA,ProteinG,sheepanti-mouseIgG,sheepanti-rabbitIgG,etc.)dependsonantibodybeingused.Youcantryotherbrands/kindsofbeads,butwehavenoinformationaboutwhatchangesyouwouldhavetomakeintheprotocolorwhatkindsofeffectsthesechangeswouldhaveontheresults.
1.Add100µlDynalmagneticbeadstomicrofugetube.Add1mlblocksolution.Setup1tubeperIP.
2.Collectthebeadsusingmagneticstand.Removesupernatant.
3.Washbeadsin1.5mlblocksolutiontwomoretimes.
4.Resuspendbeadsin250µlblocksolutionandadd10µgofantibody.
5.Incubateovernightonarotatingplatformat4°C.
6.Nextday,washbeadsasdescribedabove(3timesin1mlblocksolution).
7.Resuspendin100µlblocksolution.
BlockSolution
FinalConc.StockFor100ml
1x10xPBS10.0ml
0.5%BSA(w/v)BSA500.0mg
ddH2O90.0ml
100.0ml
III.CellSonication
1.Resuspendeachpelletof~108cellsin5mlofLB1.Rockat4°Cfor10min.Spinat1,350xgfor5minutesat4°Cinatabletopcentrifuge.
2.Resuspendeachpelletin5mlofLB2.Rockgentlyatroomtemperaturefor10min.Pelletnucleiintabletopcentrifugebyspinningat1,350xgfor5minutesat4°C.
3.Resuspendeachpelletineachtubein3mlLB3.
4.Transfercellstoahomemade“sonicationtube”(cutapolypropylene,15mlconicaltubeintotwopiecesatthe7mlmark)
5.SonicatesuspensionwithamicrotipattachedtoMisonix3000sonicator.Samplesshouldbekeptinanicewaterbathduringsonication.Todecreasefoaming,initiallysetoutputpowerto4andincreasemanuallytofinalpower(7)duringfirstburst;sonicate7cyclesof30secONand60secOFF.
[NOTE:
Youwillprobablyneedtooptimizesonicationconditions.Thesearesuggestedstartingparameters.Shearingvariesgreatlydependingoncelltype,growthconditions,quantity,volume,crosslinkingandequipment.Dependingonthepreciseexperiment,weusepowersettingsashighas9,anywherefrom3–12cyclesandvariableratiosoftimeONandtimeOFF.Ingeneral,welookforthelowestsettingsthatresultinshearedDNAthatrangesfrom100–600bpinsize.]
6.Add300µlof10%TritonX-100tosonicatedlysate.Splitintotwo1.5mlcentrifugetubes.Spinat20,000xgfor10minutesat4°Ctopelletdebris.
7.Combinesupernatantsfromthetwo1.5mlcentrifugetubesinanew15mlconicaltubeforimmunoprecipitation.
8.Save50µlofcelllysatefromeachsampleasWCEDNA.Storeat-20°C.
NOTE:
Addproteaseinhibitors(finalconcentration1x)toalllysisbuffersbeforeuse.
(DissolveoneCompleteProteaseInhibitorCocktailTablet(Roche)in1mlH2Otomakea50xsolution.Storeinaliquotsat-20°C.)
LysisBuffer1(LB1)
FinalConc.StockFor100ml
50mM1MHepes-KOH,pH7.55.0ml
140mM5MNaCl2.8ml
1mM0.5MEDTA0.2ml
10%50%glycerol20.0ml
0.5%10%NP-405.0ml
0.25%10%TritonX-1002.5ml
ddH2O64.5ml
LysisBuffer2(LB2)
FinalConc.StockFor100ml
10mMTris-HCl,pH8.01.0ml
200mM5MNaCl4.0ml
1mM0.5MEDTA0.2ml
0.5mM0.5MEGTA0.1ml
ddH2O94.7ml
LysisBuffer3(LB3)
FinalConc.StockFor100ml
10mMTris-HCl,pH8.01.0ml
100mM5MNaCl2.0ml
1mM0.5MEDTA0.2ml
0.5mM0.5MEGTA0.1ml
0.1%10%Na-Deoxycholate1.0ml
0.5%20%N-lauroylsarcosine2.5ml
ddH2O93.2ml
IV.Chromatinimmunoprecipitation
1.Add100µlantibody/magneticbeadmixfromPartII,Step7tocelllysatesfromPartIII,Step7.
2.Gentlymixovernightonrotatororrockerat4°C.
V.Wash,elution,andcross-linkreversal
Steps1through6shouldbedoneina4°Ccoldroom.
1.Pre-chillone1.5mlmicrofugetubeforeachIP.
2.TransferhalfthevolumeofanIPtoapre-chilledtube.
3.Lettubessitinmagneticstandtocollectthebeads.RemovesupernatantandaddremainingIP.Lettubessitagaininmagneticstandtocollectthebeads
4.Add1mlWashBuffer(RIPA)toeachtube.Removetubesfrommagneticstandandshakeoragitatetubegentlytoresuspendbeads.Replacetubesinmagneticstandtocollectbeads.Removesupernatant.Repeatthiswash3-7moretimes.
[NOTE:
Exactnumberofwashesdependsonqualityofantibodyandmayneedtobeoptimizedforeachantibody.]
5.Washoncewith1mlTEthatcontains50mMNaCl.
6.Spinat960xgfor3minutesat4°CandremoveanyresidualTEbuffer.
7.Add210µlofelutionbuffer.
8.Eluteat65°Cfor15minutes.Resuspendbeadsevery2minuteswithbriefvortexing.
9.Spindownbeadsat16,000xgfor1minuteatroomtemperature.
10.Remove200µlofsupernatantandtransfertonewtube.Reversecrosslink(交联逆转,即将交联打开)ofthisIPDNAbyincubatingat65°Covernight.
11.Thaw50µlofWCEreservedaftersonication,add150µl(3volumes)ofelutionbufferandmix.ReversecrosslinkofthisWCEDNAbyincubatingat65°Covernight.
WashBuffer(RIPA)
FinalConc.StockFor250ml
50mM1MHepes-KOH,pH7.612.5ml
500mM5MLiCl25.0ml
1mM0.5MEDTA0.5ml
1%10%NP-4025.0ml
0.7%10%Na-Deoxycholate17.5ml
ddH2O169.5ml
ElutionBuffer
FinalConc.StockFor100ml
50mM1MTris-HCl,pH8.05.0ml
10mM0.5MEDTA2.0ml
1%10%SDS10.0ml
ddH2O83.0ml
VI.DigestionofcellularproteinandRNA(得到纯DNA)
1.Add200µlofTEtoeachtubeofIPandWCEDNAtodiluteSDSinelutionbuffer.
2.Add8µlof10mg/mlRNaseA(0.2µg/mlfinalconcentration).
3.Mixandincubateat37°Cfor2hours.
4.Add4µlof20mg/mlproteinaseK(0.2µg/mlfinalconcentration).
5.Mixandincubateat55°Cfor2hours.
6.Add400ulphenol:
chloroform:
isoamylalcohol(P:
C:
IA)andseparatephaseswith2mlHeavyPhaselocktube(followinstructionsprovidedbyEppendorf).Optional:
WCEDNAmayremaincloudy.Ifcloudy,repeatextractiononemoretime.
7.Transferaqueouslayertonewcentrifugetubecontaining16µlof5MNaCl(200mMfinalconcentration)and1.5µlof20µg/µlglycogen(30µgtotal).
8.Add800µlEtOH.Incubatefor30minat-80°C.
9.Spinat20,000xgfor10minutesat4°CtopelletDNA.Washpelletswith500µlof80%EtOH.
10.Drypelletsandresuspendeachin70µlof10mMTris-HCl,pH8.0.
11.Save15µlofIPsampleforfuturecheckpointsorverification.
12.MeasureDNAconcentrationofIPandWCEwithNanoDrop(NanoDropTechnologies)anddiluteWCEDNAto100ng/ul
VII.T4Fill-inandblunt-endligation
A.T4polymerasebluntingofDNAends
Steps1through6shouldbekeptonice.
1.Mix2µl(200ng)WCEDNAand53µlddH2OforeachIP.
2.Aliquot55µlforeachIPsample.Keepalltubesonice.
3.Makebluntingmastermixonice:
FinalConc.Stock1xMix
1x10xT4DNApolymerasebuffer11.0µl
5ug10µg/µlBSA(NEB)0.5ul
40nM10mMeachdNTP1.0µl
1.5U3U/µlT4DNApolymerase(NEB)0.5µl
ddH2O42.0µl
55.0µl
4.Add55µlofbluntingmastermixtoallsamples.
5.Incubatefor20minutesat12°Cinthermalcycler.
6.Add11.5µlof3Msodiumacetate,pH8.0and0.5µlof20µg/µlglycogen(10µgtotal).
7.Transferreactiontopre-chilledPhaselocktubesandextract1xwith120µlP:
C:
IA(followinstructionsprovidedbyEppendorf).
8.Transferaqueouslayertonewcentrifugetubecontaining250µlEtOH.Incubatefor30minutesat-80°C.
9.Spinat20,000xgfor10minutesat4°CtopelletDNA.Washpelletswith500µlof80%EtOH.
10.Drypelletsandresuspendeachin25µlH2O.Chillonice.
B.Blunt-endligation
1.Makeligasemastermixonice(25µlperreaction):
FinalConc.Stock1xMix
1x5xligasebuffer10.0µl
2µM15µMlinkers(seeAppendix)6.7ul
200U400U/µlT4DNAligase(NEB)0.5µl
ddH2O7.8µl
55.0µl
2.Add25µlmixto25µlofsample.
3.Incubate16hoursin16°Cwaterbath.
4.Add6µlof3Msodiumacetateand130µl
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