Factors interfereing with chemiluminescence - (Mar/29/2011 )

What interferes with chemiluminescence? We transferred 100 ug equivalent of protein (from lysate) on to nitrocellulose using semi-dry transfer. Ponceau staining of the blot showed that the transfer had occurred with all samples (efficiency is still a question). When probed with primary and secondary, the chemiluminescence worked only with the standards and one of the lysates. What could be the problem? All samples have the antigen.

krithbala on Wed Mar 30 04:29:02 2011 said:

Hola I think that your chemoluminiscent substrate runs, because all the antigen samples are in the same membrane, or not?. Depending of the epitope against the ab is made or if it is made against well folded or denatured whole protein it will expose or nor the epitopes to the ab and it will reaction or not. Check how ab was made and try to see the explanation or return here for give us more details. Buena suerte

krithbala on Wed Mar 30 04:29:02 2011 said:

Could you give us a bit more detail of what you've done.

What are your standards? Is this a positive control? As for the lysates, have these samples all been run in the same gel, same membrane, and therefore incubated together? Or is this different gels and/or membranes incubated separately? What antibody concentrations are you using? How long do you incubate for? How do you block and wash?...

One of the problems could be your secondary antibody concentration being too high, as this could exhaust the ECL substrate even before you have time to measure it. Which brings me to my final question, how are you performing your detection: Film, CCD camera?

We need your help to help you, the more info you can give us the better

Are your samples degraded? how have they been stored? do you add protease inhibitors?