Excellent question, have you checked this website?

http://www.cellsignal.com/products/primary-antibodies/2365

I also have same problem, if some body knows the answer please let us know.

Thanks,

Are you sure the transfection for the plasmid itself worked? Is transcription working? If the answer is yes to both of those, then you need to ask if the protein is being translated- maybe it's misfolded and degraded starting with the tag. Maybe it's not expressed at all.

I'd check all of the above before worrying about the antibody. (The antibody could still be the problem, but with little solution- maybe the antibody is sterically hindered from reaching the his tag due to protein folding). You could also stick the his tag on the other protein terminus, if that's easier....that could avoid the potential steric problem.

Sounds like you have a lot of troubleshooting to do......

We also faced the similar problem with our His tag protein is not detected on the westerns. Although we ran the qPCR of the same cell line with His tag expression and we could easily see that our protein of interest is well over expressed. I would either change cell line or try qPCR with overexpression. By changing the cell line either you can confirm that your protein is expressed in different cell line and you have no problem with antibodies.  (This is too much i know just to make sure that if you want to stick to the same cell line, I would do the IP of His tagged protein and check wethere it would work or not) GOOD LUCK

We have seen this problem before. Almost always it is due to zero expression. Even if you see plenty of mRNA there is no guarantee that the protein is there (as Dr Jennifer Gibbons suggests above, you may have misfolding, degradation etc).

To check if there is any protein there, you could try growing a good sized batch of your cells and then after lysis, running some extract down a his-trap spin column to purify some protein that can be visualised by staining SDS PAGE gels. 

Hello

Have only worked with mouse cancer cells in regards to your problem, but this is what I suggest:

BTW: Are you trying to make a stable clone? Or do you just need transiently expressed protein for your study?

Assuming stable transfection is your goal:

No 1: find out if the gene is integrated or not. Do genomic DNA extraction and traditional PCR. Make sure your primers are suitable for the task and use wild type DNA as a negative control. 

No. 2: If the gene is there: Check for RNA expression by reverse transcription PCR. If you have the gene integrated, but no RNA expression, it could be an epigenetic problem (gene silencing). Of course, it doesn’t exclude other possible problems, but it will tell you about whether the gene integrated, but isn’t being transcribed, or transcribed, but not translated.

No. 3: You should also check for transient mRNA expression 24-48 hrs after transfections (qRT-PCR), then continue to test periodically while the cells are under antibiotics selection (up to as much as daily!!). You may find that it integrates, then disappears. If so, it could be an indication of the cells disliking the product and chops up the gene (fragmentation). In this case you may need to look for another cell line or modify your construct. Enhanced Green Fluorescent Protein (EGFP) is well liked by most cells, and when fused to your protein of interest, it can help make stable expression.

Hope this is helpful and Good luck!

Thank you all so much for your great suggestions. I am trying to make a stable cell line. As many of your suggested, I will make sure the transfection has worked by checking the expression of DNA and then move on to check the RNA expression.

I would strongly recommend to check your transformation efficiencies??? Is it really good enough for the proten to be translated and detected through WB??

You can proceed with a fresh batch of HEK293 cells. In many cases old cultures lost tranfection efficiencies and misleads expected results.

Nice discussion and I agree with above scientists. I suggest if you are not sure with your antibody, then I suggest you can try Bradford protein estimation of your transfected cells and for control you can use non-transfected cells or transfected with only plasmid. you can see a clear difference in values. Also you can raise your own antibody against your protein of interest in mice and use for your assay if you are unable to find / not available in market specific antibody against your protein of interest. 

Hope this will help you.

Regards,