Loss of signal during film exposure western blot - (Feb/23/2006 )
I've got troubles with ECL+ revelation:
after short time exposure, I obtain a weak signal, and when I prolonged the time exposure, I loose the signal!
Could someone has an explanation?
according to Pierce, this can be caused with chemi protocols by massively overloading one of your components (typically secondary antibody, if I remember correctly)
what happens is, when you go to add your detection reagent, the reaction is so strong and so driven that it burns itself out very quickly, resulting in a quick 'flash' of chemiluminesence.
have you done a western with this protocol before? perhaps stripping the blot and re-probing with more dilute secondary would help you get your data