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.

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