Ten top tips for great western blots

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1、TentoptipsforgreatwesternblotsPostedAt:December2,20101:11PM

2、PostedBy:AdamOurScientificSupportteamhaveputtogetherwhattheythinkaretentoptipsforgreatwesternblots.1.Generally,itisbettertoincubateovernightat4°Cwiththeprimaryantibody4°Covernightwillusuallyprovidemoreefficientandspec

3、ificstaining.However,manyantibodieswillalsoworkverywellwithoneortwohoursincubationatroomtemperature.2.OptimizetheantibodyconcentrationCheckthedatasheetforaguidelinestartingconcentration.Background–reducetheconcentrationofantibodyNosignal–increasetheconcentrationofantibody3.Use

4、theappropriategelpercentageforthesizeofproteinyouaredetectingProteinsize(kDa)Gelpercentage(%)4-402012-451510-7012.515-1001025-20084.LoadtheappropriateamountofsampleCelllysates,membraneandnuclearlysates:load20to30μgoftotalproteinperwell.Purifiedprotein(recombinantorendogenous):

5、load10to100ngofprotein.•Reducethisamountforhighlyexpressedproteins•Increasethisamountforlowexpressedproteins5.UsereducinganddenaturingconditionsunlessstatedontheantibodydatasheetAbcamantibodieshavebeentestedunderreducinganddenaturingconditionsonlyandwerecommendusingthesecondit

6、ionsunlessotherwisestatedonthedatasheet.6.TheblockingagentusedcanmakeadifferencetoresultsAntibodiescanbesensitivetoblockingagentsandwerecommendreviewingthedatasheettoseeifthereisanydatatoindicatetheantibodyworksbetterwitheitherBSAormilk.Generally,BSAwillgiveclearerresultsasitc

7、ontainsfewerproteinsfortheantibodytocrossreactwith.However,thisisnotalwaysthecaseandsomeantibodieswillworkbetterwithmilkasitcontainsagreatervarietyofblockingproteins.7.Don'tletthemembranedryoutatanytimeThiscanaffectantibodybindingandgivea'grey'or'black'blot.8.Checkthetransfert

8、othemembraneandqualityofthesampleusingaloadingcontrolViewourlistofloadingcontrols.9.Includeasuitablepositivecontroltoassesshowwelltheprocedureandreagentsareworking.Theremaybeinformationregardingsuggestedpositivecontrolsonthedatasheet.Ifnot,trysearchingonproteindatabasesitesand

9、PubMedforinformationontheproteinexpression.Viewourlistofselec