I carried out a dot blot for various concentrations of my cell lysate and found weak positive staining for my protein of interest at a concentration of 15-25ng. However there was absolutely no staining at higher lysate concentrations (the positive control stained even at 100ng). Can someone tell me why?
If your detetion method is based on antibodies it could be because of Hook effect. Usually this phenomenon appears with very high concentrations of analyte, and we have experienced it in some ELISA that we have performed. Good luck!
Thanks Victor. But if I'm able to detect the positive control upto a concentration of 100ng with the same antibody, I doubt that it is the Hook effect [I also know from qPCR data that the expression of the protein should be much lower than in the positive control]
Sorry I missed that part. Have you tried higher concentration of antibody? Sorry if I am not helpful. Good luck.
Again Victor, I don't think I need to increase the antibody concentration because I can get a nice, bright signal for the same concentration of positive control. Thanks for the luck!
Some proteins might be bound to their antibodies only through conformational epitopes of such proteins. Proteins tend to aggregate in high concentrations, proper folding depends on the solvent as well. could be worth considering. How does it look like on the stained gel, have you tried?
That is something to consider Jan but it doesn't make sense that only my experimental lysate is consistently not detected while the positive control is, does it? I did run a Western for the same samples and got nice bands in both samples
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