I have no idea about it, previous, when i detected this antibody, expect aim bands, background is clear. so i do not know what happened this time. 😕

I did detection again today, using same antibody, but varied protocol. yesterday, i used 5% BSA blocking, and blocked 1hr, then 1st antibody incubation overnight at 4 degree. today, i used 5% skim milk blocking, blocked overnight at 4 degree then incubated with 1st antibody 1hr at room temperature. result as following, this time, this no black spot. but I still do not know what difference for these 2 types blocking and incubation protocol. 😕

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my antibody is phospho-p38 MAPK kinase(Thr180/Tyr182) antibody. Usually, for this antibody, we used 1hr 5% BSA blocking, and incubation overnigh with primary antibody. In recent months, we used same protocol but could not get clear bands, so i changed protocol.