We have a problem when we try to detect the bands in oru membranes. Once we dye the gels we can see the bands, even after dyeing the membranes with Ponceau´s red. But after exposure to the secondary antibody and pico or femto we cannot detect almost anything.
We suppose that the secondary antibody is the responsible one, but it is a new one. We´ve even tried with different concentrations of protein.
Could anybody help us, please?
Thank you.
David
David i think, if you stain the membrane and see bands then the transfer ws properl done. Maybe the protein you are looking for is not expressed or expressed in very small amount. In this case you can try to detect the presence of other common proteins using different primary antibody. If still you dont get then increase the concentration of the protein.
Thanks. The problem is that we had signal with the same samples 3 weeks ago, using the secondary antibody when it arrived. " weeks later we had no signal at all, maybe a slightly signal after femto exposure.
Even the Actine is not there,.
I would try to increase the concentration of the secondary antibody. Just test it with actin or GAPDH after stripping. The advice of Olga Lihoadova to use a positive control could be helpful in a new try.
Is it right that you are using "SuperSignal ELISA Femto Luminol/Enhancer Solution"? In the past, I extended incubation up to three minutes. And by the way, I like employees who thought to keep Luminol/Enhancer solution at RT overnight. To change the bottles could be also helpful.
Are you using X-ray films? If you don't see a signal after 10 min incubation, I would change the secondary antibody.
what system are using for detection, could it be an inhibitor in the buffer you are using or are you incubating the secondary Ab O/N? (you should avoid doing this)
If you are using the same samples that you got bands with 3 weeks ago, your protein of interest may have degraded due to temperature issues/ freexe thaw/ protease activity. Have you fully supplemented your lysis buffer with proteases/ phosphatase inhibitors (if trying to detect a phospho protein)? Have you stored at -80C, or avoided multiple freeze thaw cycles?
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