Preparethetemplatebylinearizing25ugplasmidDNAatthe3endoftheinsert.Phenol/chloroformextract,ethanol/NaClprecipitateandresUSPendin25ulDDWTranscriptionReaction:| 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.Spin13000rpm2minAddsupernatanttonewEppendorfandethanolprecipitate2.5volethanoland0.3MNaClfinalconc..Resuspend25ulTERun3ulRNAonNortherngeltocheckthetemplate.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:Lysate200ulDDW55ulRNasin4ulAA-met6ul35Smet25ulAdd50ulreactionmixto5ulRNAIncubate30°C60min.Freeze-20oCBindingtopurifiedGEXfusionproteinsorantisera
Combine15ullysatewith5ulfusedprotein(2mg/ml)or5ulantiserain0.5mlPLCbasiclysisbuffer(seeAppendix)Incubateice1hrSwell2mgGlutathioneagarosebeads(SigmaG-4510)or2.5mgproteinAsepharose(forantisera,P-3391,Sigma)persampleinPLCbuffer/10mMDTTandresuspendbeads0.1mlPLCbufferpersample.Add0.1mlofbeadspersampleandincubateonawheelat4°Cfor1hr.Add100ulbeadsuspension/bindingreactionandplaceofthewheelat4oCfor1hrPelletsamplesinependorffor30secandremoves/ntolast50ulwithabluetip.AddicecoldPLC-lysisbufferandvortex.Repeat3x,thenaspirateallbufferfrombeadswithGilsonPipetteAdd40ulSDSsamplebufferRunonSDSPAGEgel.StainwithCoomassieanddestain.PhotographgeltocheckrecoveryofGSTproteinsorantibodyheavychainwithbeads.Amplify(AmershamRPN2106)30min,dryandautorADIographTestingprotein-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.
>>> 更多资讯详情请访问蚂蚁淘商城
