Immunoblotting traditional method

rehydratewesterninPBS-Tweenfor5min

addprimaryantibodyinaminimalamountofPBS-Tweenandincubateonarockingplatformfor1hrmin(alternativelyovernightat4CwiththetraywrappedinSaranWrap)

rinsewesterntwiceinPBS-Tweenfor5min

addsecondaryantibodyinaminimalamountofPBS-Tweenandincubateonarockingplatformfor1hr

rinsewesterntwiceinPBS-Tweenfor5min

rinsewesternonceinpH9.5Bufferfor5min

duringthisrinsepreparethenBT-BCIPAlkalinePhosphataseColorDevelopmentReagent

decantpH9.5Bufferandaddcolordevelopmentreagent(immediatelyafteritisprepared)

colordevelopmentwilloccurforabout5minandshouldbestoppedbeforethebackgroundincreasessignificantly

decantcolordevelopmentreagentandrinsetwicequicklywithPBS-Tween

washfor5mininPBS-TweenplusEDTA(100ulof0.5Mstockper10ml)

dryblotonpapertowels-blotneedstobekeptinthedarktopreventthecolorfromfading(storebetweensheetsofblottingpaper)

ImmunoblottingwithChemiluminescenceReagents

usethesameprocedureasabovebutusetheappropriatesecondaryantibodyforthechemiluminescentreagentused

addsecondaryantibodyinaminimalamountofPBS-Tweenandincubateonarockingplatformfor1hr

rinsewesternthreetimesinPBS-Tweenfor5min

useblottingpapertoremoveexcessPBS-Tweenfromtheblot

mixthechemiluminescentreagentandaddtothemembrane

iftheblotistoowetthereagentwilldiffuseandcreateexcessivebackground

exposetoX-rayfilmimmediately,themostintensesignalisobservedinthefirstfewminutes

Reprobingwesterns

westernsprobedwithchemiluminescentreagentscanbestrippedofantibodiesandreprobed

WesternStrippingBuffer(pH6.8)

SDS2g/100ml

Tris-HCL1g/100ml

preparetheabovesolution,adjustthepH,andmaketo100ml

thenaddtheB-mercaptoethanol(inthehood)

BME780ul/100ml

incubatethemembranefor30minorlongerat50CinWesternStrippingBuffer

thenwashthemembranethreetimesinPBS-Tweenfor5min

theblotisthenreadytobereprobed