Hi,
I did western and you can see my beta actin bands. I put 10 uL primary antibody for my interesting protein, but my professors said 25 uL should be. I donot understand. Wouldn't I have seen a faint band even if I put the antibody low? Is it normal to be white?
If the band is normally faint when using 25 µl of antibody, then it might be too faint to see when using only 10 µl. If the band is normally strong with 25 µl, then you should expect to see it at least faintly with 10 µl.
This should be indicating another problem. I also agree with the statements regarding the observation of even a faint band at a lower primary antibody concentration. It may be beneficial to verify the antibody first. Is it a validated and previously tested antibody? Is the expiration and storage time/condition convenient for application?
Is the transfer OK for your target, did you stain your membrane with ponceau to verify your target protein MW is at the place where it should be?
Maybe the blame is not on western but on biological conditions.
Is it possible to be an issue about the expression? Maybe your POI has downregulated, not expressed, degraded, modified, oligomerized, etc...
Good luck, İEA..
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