I am trying to determine p53 protein level in rat brain homogenate and already tried to use several primary and secondary antibodies, varying their dilution. Nevertheless, all I see are white bands on the grey film or no bands at all (however, the antibodies succesfully identify p53 in cell lysate of human HCT116 cells). Can anybody suggest, which primary and secondary antibodies (brand, cat no) and/or which dilutions can be used? And what should be the apparent p53 band "weight" on the membrane (it seems that 53 kDa is only for human p53, but the "weight" of rat and mouse p53 is a bit less (45-50 kDa))?
Hi Artem, check on Biocompare, it is a buyer's site where you can find which companies produce the antibody you need. You need to look for an antibody anti-rat p53. If you don't find one specifically raised against rat p53, check on any other that cross-react with rat (I saw one anti human that cross react with mouse and rat also). In fact, the homology between rat and human p53 protein is not high (75%) so that means that depending on where is the epitope of the antibody you have, it will only recognize the human protein and not the rat. I have just checked on Biocompare and I saw a couple that cross react. Better would be to find one anti-rat generated in mouse (if possible). Good luck.
Hi Artem, you could provide antibody against the protein yourself and conjugated it with HRP, the dilution for western blot shouldn't be very diluted.
good luck
Thank you all very much! Hope I'll find something on labome or biocompare.
I've never tried to conjugate antibody with HRP, but I'll ask my colleagues if they tried.
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