Hello,
I need help regarding my latest western blot experiment
I incubated my membrane in the primary antibody AKT overnight and then proceeded with the anti-rabbit 2nd Ab. I worked on 2 different membranes and was able to visualize a signal for 1/3. I am not sure what went wrong, but I know for a fact that the membrane that generated good and clear bands was incubated individually while the two others were incubate together in the same falcon tube. Do you know if this might be the cause of the lack of signal?
Appreciate your help!
Thanks
Could be two membranes overlapped on each other in tube. I typically twisted the two membranes in the tube to make sure they didn't have chance to overlap each other during rotation.
There might be differences regarding the primary antibody concentration or usage. Maybe the used antibody was "old" and there isn't enough left to bind to the antigens.
Use stripping buffer and try to remake the experiment with newly diluted antibodies.
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