I did a western blot yesterday, and I loaded two groups of the same samples in one gel, only separating them when I was incubating the primary antibody. The results show that my GAPDH is clean in low background, but my target gene is not clean in high background. Although they are from the same membrane and I treat them in the exact same condition.
Does anyone possibly know the reason?
Thank you!
Below are my target protein(smad2) and my GAPDH.
All I can think of is that your primary antibody against SMAD2 is not specific enough? (i.e. it is binding non-specifically to other proteins, given than you see signal all over your lanes). Are you able to try a different primary antibody against SMAD2? The best thing to do is to check the literature associated with the antibody you are buying; most of the time, companies will link you to different papers which have published data using that specific antibody.
Maybe another thing you could try before buying a different antibody is to dilute the SMAD2 antibody further? Sometimes if your antibody is too concentrated it can bind more non-specifically and lead to unexpected bands.
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