I am working with PC12 cells and. My problem is that when I perform anti alpha-synuclein Western Blots, the signals of this protein are never consistent with each other, even when I run duplicates of each sample. Is it because the protein is aggregated?In this case, what should I do?Should I sonicate my samples?Or make the lysates in a special buffer?
Maybe you can make some tests adding some Urea or Guanidinium chloride to the sample buffer. I don't know if any of them get stucked to the blotting membrane, but it may be a solution to aggregation. You should get rid off the chaotropic agent when you transfer the sample to the membrane, but I am not sure of it. I think it is worth to give it a try.
Is it happenning only when you probe the blots for alpha synuclein or is it happening when you used the lysate to probe for any other antibody. I think rather than aggression may be your samples are getting degraded. What buffers do you use to extract your lysate? Using a protease cocktail would help.
The problem appears only when I blot for alpha synuclein, the other antibodies work out fine with the same samples..... I extract the lysates in RIPA buffer with a protease inhibitor cocktail..
try glutaraldehyde cross-linking (1% in PBS for 15 minutes) before you block.
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