Hi!
When I incubate the membranes with the film 1 minute usually comes out signal but normally I need some more signal, the normal would be to incubate with another film about 3 or 5 minutes, but most often ends up losing all the signal and absolutely everything comes out blank. I thought it might be due to a high secondary antibody dilution, but it keeps happening to me. Do you have any ideas?
Thanks
Hi Sandra, It has happened to me too. I tried lowering the amount of protein as well as HRP secondary antibody concentration. Try changing your blocking buffer and make sure you use a fresh running buffer each time. Please make sure that the membrane isn't getting dried too.
Do you see a faint yellowish band on the membrane in the position you would expect your target protein to be?
Some types of ECL reagent have a very short lifetime of a few minutes once prepared by mixing the 2 solutions. If you are using one of these, consider switching to a type that lasts a long time, such as this one:
https://www.thermofisher.com/order/catalog/product/37071
Bruce Allen I haven't seen that in a long time, it did happen to me before, but not with this target protein. In this case, everything just goes blank in a few minutes.
Adam B Shapiro How do I know if my ECL is short-lived? Normally on other times with the same ECL I have managed to expose for an hour or two, so I find it strange.
OK, I was just checking to see if your signal was so strong that all the reagent was being rapidly consumed. Do you have access to a fresh lot of ECL reagents? Someone may have cross-contaminated the ECL reagents ... which virtually kills the reagents.
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