I usually transfer my gel at constant 300 mA for an hour and a half, using a 10% Tris-glycine gel. I use 15% Methanol. So after I transferred and blocked my blot, I incubated the blot with two different antibodies (seperately) overnight and tried to detect any bands. I used regular ECL and also ECL Prime and I exposed it for as long as 20 minutes. Usually, using the antibodies I use, I can detect bands using regular ECL while exposing the blot for two minutes only. I'm really confused. When I used Ponceau stain to check if my protein even transferred, I see even bands showing really good transfer. However, I cannot detect any bands using different antibodies (all fresh dilutions, nothing re-used). So I know it is not an issue with the antibody because I made a large dilution and used it for other blots and It worked for them. Any Advice?
Could you please update if these antibodies did work earlier, for the same protein, or not?
Yes , they did work earlier. I transferred four gels, two pairs. For the first pair, I can see bands. Whereas the second pair, the two blots I mentioned above, I cannot see any bands using the same kind of antibodies. All four blots were transferred in the same way ( two seperate transfers performed), yet half worked and other half (the blots I'm having issues with now) I cannot detect any bands.
Do you see some background and no bands or no signal at all?
Membrane drying in some cases could cause problems.Then u may have a lot of background with no bands.
In case u have absolutely no signal, u should worry about the secondary antibodies. Historic mistake is use of azide containing milk for HRP-labeled secondary, which kills all enzymatic activity.
Good Luck
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