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I am looking into the expression of a certain gene that is clearly overexpressed both at the gene (RT-PCR) and protein (Western blot) levels in some malignancies. However, when I test healthy controls, they are clearly positive in Western blot but completely negative in RT-PCR. I have used several different primer combinations so the reason for the negative RT-PCR is not e.g. point mutations. So, does anyone have a suggestion to how a protein may be detected while there seems to be no mRNA.
If you have a protein with slow turnover and its expression is inhibited by the activity of the protein (e.g. end-product inhibition) then the protein level could be high but the mRNA very low. If your healthy controls are not growing (e.g. confluent cells in culture) you might try reassessing the mRNA when the cells are growing more rapidly (split the cultures and assay before they are confluent). Active growth should cause the cells to make more of the mRNA & new protein as the protein will be needed to keep the cytosolic concentration near constant.
Good point!! But the cells I work with are primary cells (lymphocytes isolated directly from fresh blood). The leukemia I work on is characterized by a SLOW progression only partly due to proliferation and even more due to inhibited apoptosis.
Is endocytosis a completely unthinkable (and maybe stupid) suggestion?
Are you suggesting that the protein is being endocytosed rather than expressed in the cultured primary cells? I doubt that would be the case because on fusion of the endosome with a lysosome the proteins in the vesicle are usually degraded by the lysosomal enzymes. For the protein to enter using endosomes, it would have to either resist proteolytic degradation or prevent fusion of the endosome and lysosome. Further, the protein would have to escape from the endosome if it is to function in the cytosol. It may not be impossible for an intact protein to both survive and escape from the endocytotic pathway, but it is not common.
Sorry for not responding sooner.
The protein is indeed present in serum. A strong band is detected both in patients and in healthy controls. The molecular weight in serum is the same as seen in cell lysates. Maybe, the protein is simply bound to cell surface receptors?!? But when I do fractionation studies, the protein is detected in the cytosollic fraction. More ideas?
11 posts • Page 1 of 1
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