Optimising antibody concentration - hints and tips (Jun/02/2010 )

I have purchased a couple of new antibodies for use in Western blotting.

Just wondering if I could have a few tips on how to optimise for the best antibody concentration/incubation times/conditions to use as my initial result using the guidelines suggested by the manufacturer was pretty disappointing.

Is it just a case of repeating the blot over and over with different conditions or is there a better/faster/more efficient way?

On the same gel run sample (S) that is positive for your protein and ladder (L) side by side several times over like this :

L S L S L S L S L S

Then transfer to a membrane and ponceau stain to visualise the ladder (If you can already see the ladder you don't need to do this), cut down the middle of the ladders so that you have strips containing some ladder and some sample. You can now make several different dilutions of your antibodies and test them on the strips.