Originally Posted by meenurohini
i have been doin western blots for few months nw...since last month i started this problem of fading of the bands...the bands fade too quickly and so i cannot take the second or longer exposure...i havent changed anything in the protocol...i read that substrate depletion is possble cause for this which can be due to too much of HRP...but i havent changed the secondary concentration....can there be any other reason ??????
So you have luminescence, and it fades way too quickly.
That means you have too little of ECL reagent (could diminish with time), or too much HRP.
Too much HRP could be due to too much target protein, which then binds too much primary antibody, then too much secondary-HRP conjugate. (Since the protocol hasn't changed: probably don't have too much 1st or 2nd antibody, though dilutions or concentration would be helpful).
try reducing the amount of sample loaded (concentrations would be useful in troubleshooting).