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Protein expression but no mRNA

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Protein expression but no mRNA

Postby evamik » Fri Feb 17, 2012 8:18 am

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.
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Postby JackBean » Fri Feb 17, 2012 8:36 am

non-specific antibodies?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: Protein expression but no mRNA

Postby evamik » Tue Feb 21, 2012 7:28 am

Good point, but the detected protein is a single band of the expected size so I can not really believe that it is unspecific binding.
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Postby JackBean » Tue Feb 21, 2012 8:23 am

and did you tried your primers with some positive control?
http://www.biolib.cz/en/main/

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Re: Protein expression but no mRNA

Postby jonmoulton » Tue Feb 21, 2012 4:13 pm

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.
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Re: Protein expression but no mRNA

Postby evamik » Wed Feb 22, 2012 1:52 pm

Absolutely! And the patient sampes tested are clearly positive.
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Postby evamik » Wed Feb 22, 2012 2:01 pm

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?
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Re: Protein expression but no mRNA

Postby jonmoulton » Wed Feb 22, 2012 4:13 pm

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.
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Postby JackBean » Wed Feb 22, 2012 4:33 pm

Furthermore, if you're talking about leukocytes, the protein'd have to be present in blood serum.
http://www.biolib.cz/en/main/

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Re: Protein expression but no mRNA

Postby evamik » Thu Jun 07, 2012 8:02 am

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?
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Re: Protein expression but no mRNA

Postby jonmoulton » Thu Jun 07, 2012 2:38 pm

I suppose isotopic tracing to detect the protein origin is out of the question...
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