I have difficulty in separating the phosporylated M-MITF band and total M-MITF isoform, both of which are approximately 60kDa. I have used the cocktail of C5 clone and D5 clone, both of which are mouse monoclonal antibodies recognizing MITF for the better sensitivity in MITF detection. After exposure to SCF (10-50ng/ml) for up to 30min, M-MITF band becomes upwards shifted, however, according to the previous papers on the investigation into MITF expression in melanocyte-lineage cells and malignant melanoma cells, M-MITF band should be separated depending on the phosphorylation. The cocktail of these two monoclonal antibodies should be changed into C5 or D5 alone? Or, the protein should be harvested only from the nucleus, instead of the whole cell lysate??
Best way is to harvest the protein from the nucleus. By this method, you will get good reproducible statistically valid results. You can do it using the mouse monoclonal antibodies in the form of cocktail like C5 and D5 clone. The results will be erratic leading to frustration for not getting reproducible results.
There is some evidence available that both MITF bands are phosphorylated, as MITF contains quite a number of these sites. What you could do is using phosphatase inhibitors to prevent the dephosphorylation of the protein to see, if you can shift the pattern more to the upper band.
Since you will find phosphorylated (and thus more active) MITF in the nucleus, it is probably not a bad idea to isolate it from nucleuses. It makes more work and you will need a lot more cells, though. Using C5/D5 as a mixture or alone shouldn't make any difference, as long as the antibody works at all. I found this one of the major problems with MITF antibodies.
If you are really interested in specific phospho sites you could think about making phospho-specific MITF antibodies. There are companies available which make these.
I would like both of you for the kind comment. As Professor David tells, the cocktail of C5 and D5 will enhance the sensitivity when you perform IHC, but in the case of Western blotting, they use C5 alone to address the machinery of ubiquitination-dependent proteasome degradation of MITF. I am used to the cytoplasmic/nuclear fractioned WB in the research of Wnt/beta-catenin signal etc, I will soon try that methods and also investigate whether cocktail or C5/D5 alone is the better one.
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