I am doing western blot analysis for cultured cell histone. I am probing them with site specific modification antibody. To my dismay, i am getting result when I am developing the signal with DAB but I am not getting the same in X ray film developed by ECL. I have tried all kind of plausible combinations.. But nothing is working. Kindly suggest what can be the reason for such aberrant behaviour.
Hi Neetika,
Did you put ECL on the same membrane where you previously put DAB ?
If it is the case, then your peroxydases have probably been saturated with DAB.
If you did this on a new membrane, maybe the protein amount has to be considered.
Even if histones are quite abundant, your modifications may require a largest sample to be visible on films.
Hope it helps.
Have you tried an ECL kit with greater sensitivity? There are ECL substrates that can help with detection of low level proteins.
Additionally... with histones, I would be careful to use a membrane such as Immobilon-PSQ (PVDF) that has a reduced pore size (0.2 micron) to maximize retention of histones during the transfer, and hence maximize the ECL signal during detection.
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