I´m trying to overexpress a membrane-protein (enzyme), the problem is that I always see a big overexpression at mRNA level using qPCR, but when I use the same samples to look at protein level (either with protein or tag specific antibodies) I never see overexpression nor the tag.
I have tried using several plasmids (PCDNA 1, PCDNA 3, PCDNA 3.1 c-flag, PTRE3G...) I have sequenced all the times and there is nothing wrong with the sequence.
I have tried using several protein extraction buffers (RIPA, Hypotonic with SDS, Two different buffers with TX100...)
I am at this point totally lost, I think that subclone again and again in different plasmids is a lost of time, because at mRNA level I see overexpression with ANY plasmid, but never at protein level. I guess that there may be some postranscriptional regulation for this membrane-protein that the lines are unable to do, or that they can´t fold the protein correctly. I never see the different TAGs I have tried (V5, Histidines, C-flag)....
I have tried also different cell lines, including HEK293T.
Anyone here has been in a trouble like this? Any tip? Any idea or experiment to perform that can help me with this?
Thanks in advance.
Hello Javier
We work with multipass TM proteins in Plasmodium and upon addition of tags, have observed that their localization may change. Which means, from plasma membrane to vesicles in the cell. If you are using only the insoluble fraction to run your Western, you may miss out of the vesicular fraction.
Additionally, membrane proteins, especially those with transport or catalytic function are regulated in terms of their surface expression. The cell may not translate just because there is more mRNA, just saying. In the event of a stimulus that demands higher surface expression of the said TM protein, we observe increased surface localization of the protein and increased expression detected via Western blotting.
Coming to your protein, have you tried to observe the localization of the same via Immunofluorescence (IF) microscopy? Also, it may be possible that the fusion protein was misfolded. IF can help you in this regard since you look at whole fixed cells without segregating compartments. If you find your proteins in vesicles, or punctuate forms then you know at least that the protein is there. If nothing can be seen in IFA, this may indicate some post-transcriptional modification, that may interfere with expression.
Hope this helps
Best
Have you ever tried to express your protein in a cell-free expression system? This can get around the problem of proteins that can't be expressed because they are cytotoxic.
@Aparajita thanks for your recommendations.
I´m not using insoluble fraction to run the WB, just whole cell lysate after 48 hrs of transfection, the qPCR shows 10 cycles of difference between empty plasmids vs my plasmids, but nothing visible at WB.
Your idea about using IF microscopy is so useful (I can do it but I have not though about it!). Is the best way to confirm if there is traduction or not, if there is not traduction I am sure that this multipass TM protein is so difficult to fold.
Thanks a lot!
@Adam thanks for your reply.
No, I have not tried to express it on cell-free expression system because I am interested on the effect of this protein on cell biology. However, I think that the protein is not cytotoxic because with the transfection we don´t observe cell death, the problem is that not even one single tag is observed, and even if the transfection / expression ratio is low, it should be possible to see the tags or a little overexpression, even more when at qPCR there are 10 cycles of difference between empty plasmids vs my plasmids,
Hi Javier
Have you checked your transfection efficiency ? It might be that you have protein but below the detection limit of western blot. You can try also to concentrate your sample and test it in western.
Hello Javier,
Are you isolating membrane proteins to compare the levels in WB? If you are and still no luck, I would recommend looking for other protocols for cell membrane extraction. That would be your best bet.
Also, if your protein is a tightly regulated protein in a system there is a possibility that it is degraded post-translation. I have observed a similar trend in-vivo in Tg mice over expressing the gene of our interest. However, we did not find this during our in-vitro studies. In that case you can look for UPR (ER stress) protein changes.
Best of luck,
Esha
Hi Javier
I would think that the problem is technical , i.e. of determination of your protein. If you have mRNA and it is known that the protein is expressed in the membrane it should be there (and it doe snot matter much if you use one or another method of extraction) According to this, I wonder whether it may be any issue related with extraction (i.e. your "sample" does not contain your protein because when you centrifuge it goes to another fraction) and/or problems with Ab or Western blot. Concerning the first possibility you say that you "use the same sample" Samples to check for mRNA and for protein must be different.
Concerning the second possibilty you may run positive controls. In this case positive controls will help knowing whether Ab or Western work properly
Thanks all for your replies, I´m going to check if there is UPR protein changes, together with the IF I think it should offer some clue about the problem.
@Rafael Thanks for your reply! When I say that I use the same sample for WB and qPCR I mean duplicates of the same treated / control cells but extracted with different methods (Tripure for mRNA and different buffers for WB). With the WB I think that there is no problem since all my others WB (including overexpression of RAS as a positive control of transfection) are ok. The ABs were tested with basal cells, the tag one is a really good one, and the protein one is the best one that we have tried....it´s a difficult case :/
Dear Javier,
as you probably know, the amount of RNA does not necessarily reflect the amount of protein produced. Do you know about the biological half life of your protein? We once detected expression of a short-lived protein by Western blot only upon addition of a proteasome inhibitor after transfection of the cells. Maybe it's worth to try? Or try pulse chase biosynthetic labelling and immunoprecipitation?
Good luck, Ingrid
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