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same proteins, same antibodies, different results - (Mar/03/2007 )

Hi to everyone,
I've a big problem with westerrn blot on PBMC.
Almost 10 months ago I performed an experiment for detection of GSS on PBMC protein extracts and the results were very good.
Some weeks ago I tried to repeat the same experiment on the same proteins but the Xray film show a strange bands (in the first experiment the bands were perfect such as a book of protocol), more background, several unspecific bands that I cannot remove, the housekeeping gene seems to show that I've loaded not the same quantity of protein (40µg). So I've repeated the BSA-bradford method and the concentration are correct.
I thought that the cause could be not fresh protein, and I've used others new. The antibodies are the same.
I do not understand what is happened to my hands, perfect Xray film 10 months ago and a disaster now.
I hope someone can help me. sad.gif


have you re-made your buffers fresh? it could be that there was a mistake in the blocking formula, or one of the wash buffers

are you using fresh aliquots of your antibody?

for this to happen, something must have degraded, and you have ruled out your protein so it must be something else smile.gif