Hi, I performed western blotting for the 8 times. In two of them I used wet-transfer and the rest of them assayed with semi-dry transfer. I checked my buffers, systems, gels and everything. I used two different primary antibodies which one of them is surely working. In the first 5 assay, I observed signal from the antibody that we know it is working. after we took images, I stained the membrane and observed protein bands on it. but now I can not observe any signal neither of them. and there is any protein bands on membrane. what could go wrong?
If you do not detect any protein on your membrane after the staining procedure (I guess for your experiment, using ponceau S), it is most probably the transfer issue you are facing...Putting aside the antibody verification, if there is not any protein on your blot but you have them on your gel after SDS-PAGE, this must be investigated during the transfer process. How about your ladder, can you see them on your PVDF membrane? Your PVDF membrane conditioning (pre-wetted with MeOH) protocol has been performed properly?... Applied voltages and transfer protocols are valid and buffers are prepared fresh?... Do you observe this in both semi and wet-transfer platforms?
After blotting has been completed, you may check the gel again and put a secondary PVDF membrane during transfer to see if any protein either passes through the membrane or remains in the gel...This will clarify if this is a transfer issue...
If I am reading this correctly, you were able to get images from the membranes, then you stained the membrane and saw protein, and then you tried to reimage the membranes and didn't get signal the second time? If that is the case, you should be aware that ECL loses its signal over time. It only takes about an hour after application for the signal to have completely disappeared. Also I'm fairly certain that staining the membrane would remove any active ECL.
Fluorescent probes do last longer if they're kept in the dark. For those blots, you can typically image them for several days after finishing. I confess I do not know if staining the membrane would have any effect on that techniques or not, but I wouldn't be surprised if it suppressed it. I have not tried reapplying ECL to a blot that I have already imaged unless I have completely reprobed it first, but hypothecially rinsing the membrane in TBST and then reapplying ECL should work to get signal back. I never really bother staining membranes myself, but I wonder if the stain itself could be blocking any attempts to reimage using chemiluminescence.
ECL fading is particularly true if you're using any of the formulas designed for digital imaging. The formulas designed for film last a bit longer but produce less light immediately, the ones designed for digital imaging produce more light quickly but fade faster.
I also have better results with wet transfer compared to semi-dry but if the membrane initially gave you results and now it's not, that is probably not the problem.
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