Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

blinking results in western blot - (Oct/10/2007 )

Hi everyone!
My experience with western blot is about 3 years. Every time everything is working well! But now I have a mysterious problem. Some times I can’t see any bands. Some times I made western blot and I have got no signal, than I without stripping made the same western blot (with the same Ab’s and reagents) with the same membrane and I see the signals. I don’t understand the reason of this. Can anybody advise me anything?
What is may be wrong?
membrane is blocking in milk +4C Over night
incubation with 1st ab"s 1 hour RT
3x5 min washes in TBSt
incubation with secondary Ab's 1 hour RT
3x5 min washes in TBSt
ECL kit
develop the film
Thank you in advance.


Your first antibody incubation time is very short with only one hour at RT, perhaps you get better results if you give your antibody longer time to bind. I always incubate over night at 4°C and block one hour at RT. Everything else semes to be o.k. Is your ECL-kit too old? Or your membrane? Try to use everything as new and fresh as possible.


biomaus, thank you very much for your answer
It turned out that problem was in milk... Now everything is ok.