REAGENTS
ECLWesternblottingkit(AmershamLifeScience;cat#RPN2108):containssecondantibodiesforbothmouseandrabbit,substrateandmilkblocker(themilkblockerisnotnormallyusedwhenusingruminantsamples).
HybondECLnitrocellulosemembrane(AmershamLifeScience;cat#RPN2020D)
KodakX-OMAT(XAR-5,18X24cm;cat#8532665)
10XTBS
12.11gTris-base(100mM)
87.66gNaCl(1500mM)
1literddH2O
AdjustpH=7.6
Washingbuffer(TBS-T):100ml10XTBS+900mlddH2O+1mlTween-20.
Blockingbuffer:TBS-T+1.5%gelatin
IncubationbufferI(forfirstantibody):TBS+1.5%gelatin
IncubationbufferII(forsecondantibody)=blockingbuffer(i.e.,TBS-T+1.5%gelatin)
PROCEDURES
1-Immediatelyafterremovalfromtheblottingapparatus,placemembranesintoblockingbufferfor2hours.Asmallplasticgelboxisasuitablecontainer.Thisandallotherincubationstepsareperformedatroomtemperatureandintherockerplatform.
2-Washmembraneinwashingbuffer:rinse2timesverybriefly,incubatefor15minutes,thenrepeat2xat5minuteseach.Usealotofbuffer.
3-Transferthemembranetoalidof96-wellmicrotiterplateorsimilarlowvolumecontainer.IncubatewithfirstantibodyusingrecommenceddilutioninTBS+1.5%gelatinduring2hours.Approximately10mlofdilutedantibodyshowedtobesufficient.
4-Transferthemembraneintogelboxandrepeatstep#2.
5-Transferthemembranebacktothesmallcontainerandincubatewithanti-mouseoranti-rabbitIgGhorseADIshperoxidasediluted1:8000inTBS-T+1.5%gelatinfor1hour.
6-Repeatstep#4(wash)
7-PrepareECLsolutionfordetection:mixequalvolumeofECLreagent1and2,(1:1)withfinalvolumeregardingof0.125ml/cm2(foraminigel-4mlofeachreagent).
8-Removeexcessbufferfromthemembranebydrainingthemembraneoverapieceoffoldedkimwipepaperandbrieflytouchingtheedgeofthemembranetothepaper.
9-AddECLsolutionandincubatefor1minute.
10-Repeatstep#8(drainexcessofsubstrate)
11-Placemembranedownonseranwrap,removebubblesandwrapmembranecompletely.
Avoidexcessamountsofseranwrap.
12-Tapemembranetotheinsidefilmcassette.
13-Inthedark,add1sheetofx-rayfilmtothecassette.Exposemembranetofilm.Itwillprobablybenecessarytodoseveraldifferentexposurestofindoutthebestexposure.
14-Developthefilm.
