I'm trying to detect p53 in cytoplasm in skin fibroblast by using Western Blotting and Immunocytochemistry. I could detect p53 in nuclear but not in cytoplasm. I know that p53 rapidly undergoes proteasome degradation in cytoplasm. I need to find out how much p53 is produced in cytoplasm.
You can try proteasome inhibitors like MG132 or lavacystin to prevent protein degradation. I am not sure about WB, I think that immunolocalization is more suitable way in this case, if you have a good antibody. Good luck!
You can also see our paper : I. Ferecatu et al. (2009) " Mitochondrial localization of the low level p53 protein in proliferative cells". Biochem. Biophys. Res. Commun. 387, 772–777.
Well as mentioned here p53 is mainly nuclear so not much you can do about that. As far as I know all the proteins are synthesized in the cytoplasm so if you need the rate of translation that can be done with pulse labeling and then maybe fractionation of the cells to cytoplasmatic and Nuclear fraction. That way you might get an enrichment for newly synthesized p53 that might be more in the cytoplasm. If you want to manipulate the p53 gene so it would be in the cytoplasm you can mutate the NLS , this has been previously done in numerous publications. and of course stressing the cells increases the chance of seeing p53 in the cytoplasma as in general there will be more of it.
Hope it helps
Peter
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