Resolved my protein (envelope glycoprotein ~ 65 kDa) by 12% SDS-PAGE and transferred to nitrocellulose paper for western blotting. Have detected the same protein before, and it worked, but the last few blots have been staining the same protein white. Why may this be so? I have included pictures...
If this is western blotting (your images do not make this clear), this can happen when your signal is so intense that all ECL reagent in the immediate vicinity is exhausted before you even put the film down. In essence, there's so much protein that it generates an intense burst of light and then nothing.
The fact that you're seeing a lot more background (assuming I'm interpreting the images correctly) suggests these are longer exposures.
Alternatively, is the antibody a different batch?
If this is western blotting (your images do not make this clear), this can happen when your signal is so intense that all ECL reagent in the immediate vicinity is exhausted before you even put the film down. In essence, there's so much protein that it generates an intense burst of light and then nothing.
The fact that you're seeing a lot more background (assuming I'm interpreting the images correctly) suggests these are longer exposures.
Alternatively, is the antibody a different batch?
It looks like NBT/BCIP staining to me and like primary antibody stopped working. So, your protein of interest works like effective block against unspecific background NBT/BCIP staining because you have lot protein in one spot. Try making fresh antibody solution.
Hi Jason, is this a blot developed with NBT/BCIP as Marija observed? That can make a huge difference in explaining the negative bands.
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