Hello
I am currently trying to get a specific band for CaM (17kDa), according to Cell Signaling; the antibody which the lab bought, I should use: 1:1000 for primary dilution in 5% BSA in TBST all night... sooo, no specific band!! But tons of non specific binding!!
I also tried the antibody in 5% skim milk in TBST, just TBST, PBS+BSA 3% (the one in the pic, sugested by my boss), but no luck so far.
Recently, we bought the goat serum for blocking the membrane... I am thinking of using it...
I use a 15% SDS-PAGE, with a PVDF membrane, using a semi-dry transfer.
Any ideas, are welcome!
Also, as anybody have problems with antibodies from this company? as far as I know, they are the BEST company for WB antibodies... not like Santa Cruz... unless I just got a bad antibody :(
Thanks! Gracias :)
I think 5% skim milk is fine as blocking agent. you can rather try with higher gel % and lower dilution of antibody.
Hello Nancy,
maybe the blotting is a problem, looks like the protein amount on the membrane in the below 20 kDa area is not to great. Maybe you could try a Ponceau staining to test if enough protein transfers to the membrane in this area. But this would of course not explain the many bands in the higher areas. I do not think that the blocking is the problem. Maybe rather the sample, that CaM despite of the SDS still bind to a his binding partners. Fresh SDS is maybe worth a try. And of course it is always possible that the antibody turned somehow bad.
Good luck,
Carsten
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