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Antibodies don't work

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#1 DarwinDuck



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Posted 15 March 2011 - 10:09 AM

Hello everyone,

I have a big problem with Western blot in getting results. I assume that my antibodies stopped working (primary and second).
I work with primary chondrocytic cultures, by which i isolate the cell extract. I use 25μg of proteins for running in a biorad device.
I transfer in invitrogen device and my ponseu staining is excellant. I also paint with Coomassie my gel to be sure of complete transfer. I block O/N with 5% Milk
Anyway, I didn't change anything of the technique and i suppose that until ponseu staining, things work fine!
The problem begins when i probe my membranes...I use antibodies that have worked in the past (about 1 month before) and new antibodies too. None of them work! I made new second antibodies and nothing yet. I made smaller dilution of the new antibodies, still nothing. I have also probed my membranes with actin, but nothing!! My samples and most of antibodies used to work in the past but now i don't know where is the problem..I stained again my membranes with ponsue and i saw that most of the proteins have gone, without getting any resulta!!!
I im in a big hurry, because i am trying to finish my PhD and this is really frustrating!!!Any halp????

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