RIPA Buffer,5X_实验搜索
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- Preparethetemplatebylinearizing25ugplasmidDNAatthe3'endoftheinsert.Phenol/chloroformextract,ethanol/NaClprecipitateandresUSPendin25ulDDW
- TranscriptionReaction:
| Reagent | amt/reaction |
| 5xtranscriptionbuffer | 20ul |
| 10xDDT | 10ul |
| DDW | 40ul |
| rNTP | 20ul |
| RNasin | 0.5ul |
| T7RNApolymerase | 1ul |
Addto5ugDNAtemplate.Mixreaction,addingenzyme(T7)last.Incubate37°C90min
- Addequalvolumephenol/chloroform(100ul).Vortex.Spin13000rpm2min
- AddsupernatanttonewEppendorfandethanolprecipitate2.5volethanoland0.3MNaClfinalconc..
- Resuspend25ulTE
- Run3ulRNAonNortherngeltocheckthetemplate.
Northern
| - | 1.5gagarosein130mlDDW.Boiltodissolveagaroseandadd15ml10xMOPS.Coolandadd15mlformaldehyde.Add3ulRNAand7ulSamplebuffer.Samplebuffer(90ultotal)=50uldeionizedformamide,20ulformaldehyde,10ul10xMOPSand10ul400ug/mlEthidiumBromide |
| - | HeatRNA65oC10min |
| - | Runat100v2hrsin1xMOPS.Thesampleshouldcontain0.2-1.0ugRNA. |
Translation
WeusePromegarabbitreticulocytelysate.S35methionineisDuPonttranslationgrade(5mCi/mmol)
- HeatRNA60°C5min,iceandpreparereactioncocktail.Thelysatecanberefrozenonce.Onlyprepareacocktailwiththeamountrequired.
- Forexample:
- Lysate200ul
- DDW55ul
- RNasin4ul
- AA-met6ul
- 35Smet25ul
- Add50ulreactionmixto5ulRNA
- Incubate30°C60min.Freeze-20oC
BindingtopurifiedGEXfusionproteinsorantisera
- Combine15ullysatewith5ulfusedprotein(2mg/ml)or5ulantiserain0.5mlPLCbasiclysisbuffer(seeAppendix)
- Incubateice1hr
- Swell2mgGlutathioneagarosebeads(SigmaG-4510)or2.5mgproteinAsepharose(forantisera,P-3391,Sigma)persampleinPLCbuffer/10mMDTTandresuspendbeads0.1mlPLCbufferpersample.Add0.1mlofbeadspersampleandincubateonawheelat4°Cfor1hr.
- Add100ulbeadsuspension/bindingreactionandplaceofthewheelat4oCfor1hr
- Pelletsamplesinependorffor30secandremoves/ntolast50ulwithabluetip.AddicecoldPLC-lysisbufferandvortex.
- Repeat3x,thenaspirateallbufferfrombeadswithGilsonPipette
- Add40ulSDSsamplebuffer
- RunonSDSPAGEgel.
- StainwithCoomassieanddestain.PhotographgeltocheckrecoveryofGSTproteinsorantibodyheavychainwithbeads.
- Amplify(AmershamRPN2106)30min,dryandautorADIograph
Testingprotein-proteininteractionswithinvitrotranscribedandtranslatedproteins.
Thetwoproteinsshouldbetranslatedsuchthatoneislabelledwith35Smethionineandoneisunlabelled.IfbindingistobeassayedbyImmunoprecipitation,thenthe"bait"proteintowhichtheprecipitatingantibodyisdirectedshouldbeunlabelledandthe"target"proteinlabelled.HavingthetwoproteinslabelledispossIBLe,butprobablybestavoidedtokeeptheamountoflabeldown,andincaseswhere"target"proteinand"bait"proteinsarethesamesize.Acontrolwithvectoronlyas"bait"shouldbeusedtoshownon-specificbindingoflabelledproteinstothebeads.Ifbackgroundisaproblem,thenblockingagentssuchas5%skimmilkpowderor0.8%BSAcouldbeused.PreclearingtheimmunoprecipitationwithproteinA/Gsepharoseisalsoapossibility.
- PrepareproteinsseparatelybyinvitrotranscriptionandtranslationaccordingtothestandardPromegaTNTreticulocyteprotocol.Adjusttotalreactionvolumesothereis12.5ulofeachlysateperbindingassay.
- Add12.5ulofeachproteinlysatetoaneppendorfandmixbypipetting.Allowproteinstoassociatefor30minutesat4°C.
- Add200ulofcoldimmunoprecipitationbuffer.SuitablebuffersincludebasicPLC(withorwithoutEGTA),RIPAbuffer(withorwithoutSDS)andPBSwith0.01%lubrol.Choiceofbufferisdependantonthestrengthofinteraction.ProbablythegentlestconditionsarePBSwithlubrol.Beawarethatsomeprotein-proteininteractionsmaybedependantonmetalionssuchaszinc,andtheseshouldbesupplementedwhereappropriate.
- Addantisera(5-10ulcrudesera)and2.5mgproteinA/Gsepharose.Incubateovernightat4°Cwithgentleagitation.
- Spindownbeadsandwashthreetimesin1mlofimmunoprecipitationbuffer.Thefirstwashshouldbecarriedoutinthehoodandtheradioactivewastediscardedappropriately.
- Resuspendbeadsin30ulSDSsamplebufferandrun20ulonapolyacrylamidegel.Drygeldownandautoradiograph.
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