If the epitope of your antibody is the very end of the C-terminus, it is possible that the antibody doesn't recognize your protein when it's concatenated with dsRed. (it happened to us with an mCherry concatenated protein....)

If your DNA construct is definitely ok (reading frame ok, no unwanted STOP codons) you should check for DNA/RNA secondary structure at the boundary between the dsRED and your target protein or at the 5' end of your target protein - maybe it forms hairpins which could lead to premature termination of translation. If this could be the case you could have that fragment synthesised with wobble-position modifications to get rid of the secondary structures while the aa-sequence remains unchanged.

There is also the possibility that your target protein is processed e.g. if an N-terminal signal sequence is present it may just be cleaved off the dsRED - which would in the case of the fusion protein cleave the C-terminal part (your protein of interest) and leaving you with the target protein running exactly at the size of its untagged endogenous counterpart...

Thank for your answer. My protein is cloned with C-terminal-DsRED so I do not think I have problems with the N-terminal signal sequence, but maybe with the secondary structure in RNA, but how can I check this?, I have already made another construction with another gene bu t with Cterminal gfp and it is ok I do not have troubles.

If your DsRED is at the C-terminus even the secondary structure cannot be the problem - if the translation would be terminated by it you would loose the DsRED, not the target protein. So I would not consider the RNA to be the problem. Since you get the DsRed signal, the RNA is definitely translated. Could still be some cleavage site in your protein of interest, maybe check it with e.g. web.expasy.org/peptide_cutter/ for potential cleavage sites. If you add another tag to your protein at the N-terminus e.g. a FLAG or MYC tag, you could discrimintate a potential cleaved form from the endogenous protein.

If the epitope of your antibody is the very end of the C-terminus, it is possible that the antibody doesn't recognize your protein when it's concatenated with dsRed. (it happened to us with an mCherry concatenated protein....)

Hi Rosa,

check if your antibody is specific for the c-teminal sequence. The DsRed may have changed the epitope recognized by the antiboby. As a double check, probe your membrane with an anti-DsRed antibody. You should get the fusion-protein band or, if Daniel is right, the cleaved DsRed band.

Hi Rosa,

As Anna and Tommaso mentioned before, your problem may be due to epitop masking by DsRed, assuming that the antibody against your protein of interest is directed to its c-terminus. You could use anti-DsRed (general anti-GFP antibodies should work for that) and look for accordingly higher molecular weight band that represents your fusion protein. Another problem may be multimerization of DsRed. Some older versions of DsRed have a tendency to do that and it may lead to epitope masking. Is shouldn't be a problem when running denaturing SDS-PAGE but things happen. If thats the case you may try running SDS-PAGE with addition of urea.

Good luck!

Before you go to the trouble of changing your construct or creating a new one, try blotting with a different antibody - as Anna said, occasionally a fusion will disrupt what is normally a good epitope. Since that's the simplest solution, give a try and hope it solves the problem.

Also, I presume you are quite sure that it is being expressed via confocal? If you cannot confirm via WB you may want to do another transfection and check again, to make sure you are certainly dealing with something that is translated but not appearing on your Western.

Hi,

apart from antibody not able to recognize the constructed the protein (as many suggested and i do agree with them); you might want to consider that the constructed protein might be in the membrane fraction which can get lost as cell debris after cell lysis.

to avoid this, check with a western blot on membrane and compare with cytosolic fraction of the cell or simpler check with a florescence spectroscopy of lysed cell fractions before western blot (will save a lot of time) if the protein structure is still in tact after lysis.

if this doesnt work - try lysing the cell with gentler reagents and see periodically under a crude flour spec. you can map out where the problem is.

all the best.

Hello,

Do you have a positive control on your western blot? When you incubate you WB with anti-RFP do you see RFP band with the RFP size?

Is the gene cloned in frame? If you have an frameshift mutation, you might see the RFP aloneon the confocal, and you would also get an RFP signal on your PCR.

Are your primers directed to your gene or RFP alone?

Can you make a primer that would amplify the fusion protein?

I hope it helps,

good luck, Claudia.

Another question, did you tried to fix your cells and have a look on the microscope to see if proteins are well expressed?

I would agree with Claudia that it would be good to use an anti-RFP in parallel to see what is happening from a RFP perspective. We have made a number of EGFP c-terminal fusions that have been analyses via conventional fluorescent microscopy for some consructs we can definitely see the EGFP fusion but can't detect it on a Western. So it seems there's a cutoff there that's hard to quantifiy. So if you see much less RFP in the fusion transfections compared to RFP along it may not be in a detectable range on a western. A polyclonal antibody to either RFP or your protein might help solve this. Also a supersentitive chemiluminencent substrate might help - though increasing sensitivity might give you more problems! There's also options like immunoprecipitation.

Thanks for the answers. I am trying the new antibosdy anti-DsRed. I added a file with pictures of my transfected HeLas and we can see red cells.