High mRNA but no protein expression - (Jan/13/2011 )
i have performed RT PCR and i have found for my gene that the mRNA levels are very high.the problem is that the protein is not expressed in the cells!!!any ideas why?the sequencing is ok,the western blots do not detect protein but the RT PCr gave a high amount of mRNA.thank you
Antibdy wasn't working? protein is expressed but at levels below detection limits? mRNA is pooled for later use? your PCR is picking up genomic DNA?... there could be any number of reasons.
Thank you for your help,
i have two probes:one whole receptor and the receptor with a depleted sequence!in western blots i can detect the whole receptor but not the depletion!in LSM microscopy i can see the receptor(tagged with GFP) on the membrane but the depleted Receptor is all over the cytosol the GFP stain!depletion seems so to be fast degraded but RT PCR gives me high amounts of this mRNA even higher from the normal receptor.any ideas?
Assuming the cytoplasmic GFP stain is specific for your shortened protein, quick degradation (at least for the fusion protein) should not be a problem. Are you sure the deletion will not eliminate the antibody's epitope? If an antibody against GFP is available, you could try to detect the GFP fusion protein by western blotting using antibodies against both GFP and your protein in parallel. Another option to check your antibody would be immunocytochemistry.
I have GFP Ab.i am doing all my western blots with that,the receptor and the control receptors(normal and depleted) tagged with GFP are detectable but not the depleted one,GFP band is also there(26 kDa) but no protein.I have tagged also the receptor with flag and did the IF and i had no signals for the depleted! i am investigating the possibility that this depleted part plays any role with the expression of the protein.this is why i did the RT PCR to see the mRNA levels!it is a bit complicated.Actually my question is:mRNA is high but the protein is no expressed or better fast degraded,what can i say actually about the depleted part?is it implicated in these 2 things? total lost here!thanks for all your help
Have you checked that the depleted one is actually in-frame with the GFP - it could be that there is a frameshift in the cloned sequence, so that the GFP is being expressed properly, but the insert isn't it would also explain why it can be detected by PCR as this is not dependent on frame at all, but not be western/IF.
The insert and the gfp are in the same frame shift,i have checked the sequence and it is ok.
In my opinion, rapid degradation of protein or mRNA is not the reason for failure of detection on protein level. I that was the case, you also wouldn't detected GFP.
Is the GFP fused to the N- or C-terminus of your receptor?
i have read that in some cases a fast degraded protein has higher levels of mRNA cause the cell is trying to compensate the difference!The GFP ic at the C-terminus fused so as to the control receptors!
What part of your receptor is deleted in your depletion version (NT,CT,middle)?