SemiQuantitative Measurement of Proteins by Dot Blotting

Purpose...Concentrationofproteinsinacrudepreparations(suchasculturesup)canbeestimatedsemi-quantitativelybyusing"DotBlot"methodifyouhavebothpurifiedproteinandspecificantibodyagainstit.MaterialsTBS:20mMTris-HCl,150mMNaCl,pH7.5TBS-T:0.05%Tween20inTBSBSA/TBS-T:0.1%BSAinTBS-TNitrocellulosemembrane(Bio-Rad,Trans-Blot™etc)Procedure1.Havenitrocellulosemembrane(NOTPVDF!)ready,drawgridbypenciltoindicatetheregionyouaregoingtoblot(seebelow).2.Makeserialdilutionsofpurifiedprotein.ForquantificationofIgG,preparethefollowingsolutions0,0.03,0.1,0.3,1,3,10,30,100ng/µlofmouseIgGinTBS3.Makeserialdilutionsofyourunknownsample,suchasx1,x3,x10,andx30dilutionsforhybridomaculturesup.Makecontroldilutions(freshculturemediumatthesamedilutions)aswell.4.Usingnarrow-mouthpipettip,spot1µlofsamplesontothenitrocellulosemembraneatthecenterofthegrid.Minimizetheareathatthesolutionpenetrate(usually3-4mmdiam.)byapplyingitlittlebylittleover2-3timesofstrokeofpipet.

5.Letthemembranedry.6.Blocknonspecificsitesbysoakingin5%BSAinTBS-T(0.5-1h,RT).Use10cmPetriDishforreactionchamber.7.Incubatewithprimaryantibody(0.1-10µg/mlforpurifiedAb,1/1000to1/100000dilforantisera,x1tox1/10forhybridomasupernatant)dissolvedinBSA/TBS-Tfor30minatRT.8.WashthreetimeswithTBS-T(5minx3)9.IncubatewithsecondaryantibodyconjugatedwithHRP(foroptimumdilution,followthemanufacturer"srecommendation)for30minatRT.10.WashthreetimeswithTBS-T(15minx1,5minx2),thenoncewithTBS(5min).11.IncubatewithECLreagent(1:1mixtureofSolution1and2,oryoucandilutethemixedreagent2to10-foldwithwaterifthesignalwastoostrong)for1min,coverwithSaran-wrap(removeexcessivesolutionfromthesurface),andexposeX-rayfilminthedarkroom.Tryseveraldifferentlengthofexposure.12.Comparethesignalfromyourunknownsampletothatofstandardandestimatetheconcentration.Keywordsproteinquantitation,nitrocellulose,antibody,chemiluminescence,peroxidase