Hello,
We are currently measuring concentrations of protein kinase A (PKA) and its phosphorylated from (p-PKA) in sub regions of the hippocampus using α-tubulin as loading control. We are using semi-dry blotting (with Bio-rad's trans-blot turbo system)
This worked fine until recently when we started seeing at first vastly different intensity of signal (both band of interest and loading control) between lanes which naturally at first we attributed to problems with loading of the samples. However the next day on retrial the signal was not only varied between lanes but also mostly gone all together with exception of two samples (their lanes were far apart) and now we are just getting completely empty membranes except for the ladder markers.
The same antibodies, parameters and blotting protocols etc. were used as before during successful blotting.
At first we used the same running buffer etc. but even after preparing every possible solution anew the problem persists.
Testing with the same samples that worked before also produce empty membranes now.
Also, probably not relevant but before we started getting no signal whatsoever, for some reason two samples taken from two animals would always appear stronger than the rest (almost normal) even if the samples from different hippocampal regions were tested.
Did anyone encounter such problem before and if so how did you deal with it?
Any suggestions will be appreciated,
KS
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