We had a similar problem in our lab: there were no good antibodies against our protein of interest and despite high mRNA expression (qRT-PCR), we did not see the protein on our westerns (low sensitivity of antibodies). So we used a chimeric construct (protein+GFP) and detected the protein with an anti-GFP antibody on westerns. That worked! You could try this after having ruled out other western blot problems.

"Protein present..."

or

"..but by Western Blot there is no overexpression of the proteins.."

what the question?

Hi Ana Rita,

have you checked that your cell lysis/Western blotting protocol is working? For example, have you blotted for a housekeeper, such as beta actin? Or have you run a coomassie to see amounts of protein you have?

Also, are you running a positive control for your proteins (i.e. a lysate that definitely has your proteins of interest, such as those you can buy commercially)?

Are your constructs in vectors with tags (i.e. EGFP, YFP, mCherry, Xpress)? If so, are you immunoblotting with antibodies against the tag or the protein of interest? In my experience, if your protein is tagged it is easier to blot with an antibody against the tag.

Also, how big are your proteins?

All best,

Pela

Pelagia I used cycloA to control and it's ok.

The supplier did send me a positive control and I was able to see it on the film. But not my proteins. I have ordered more positive controls (still waiting). 

My proteins do not have any tags and they are ~ 8-10KDa. 

Thanks

:)

 Hi Ana ,

I agree with Pela, You must check house keeping genes like alpha tubulin, beta actin or GAPDH to see if you have done the western blot correctly. After onwards, you can use positive control samples.

Best,

Khalid Khan

Hi, Ana,

for your problem, several aspects that you should pay attention:

first, the plasmids transfection rate, which transfection reagents  that you used?

second, the transfer membrane condition that you should need further to determine or optimize, because the protein molecular weight of you interested  is too low, so you should pay attention to the details such as when running the SDS-PAGE, monitoring the band position according to the protein ruler; your protein is easy passing to the PVDF or NC membrane, maybe this is the key to your problem.

third, detection the housekeeping genes can prove the quality of your cell lysate, however, this is not the important for your interested protein, the cell housekeeping genes can be detected if you according to the lysis protocol correctly . after all, your interested protein was expressed or not  in your transient transfection is determined by the mount of plasmid that trasfected into the cell. I think, the vector and the promotor in the vector are good.

last, if above all righ but the protein is still not expression, please checking the primers that you designed or change the other vectors.

best regards,

Guangcheng Xie

Hi, Ana.

Perhaps you can check the expression of the mRNA of your proteins by RT-qPCR. If there's no mRNA of your desired proteins, maybe it's because silecing via promoter methylation or because of problems with transcription.

If there are enough mRNA levels but still no protein, it could indicate silecing via miRNAs. In miRTarBase you can check the different miRNAs that target your proteins' mRNA.

Good luck!

Fernando F. López Calderón.

Hi,

A couple of questions...

1) What plasmids are you using to overexpress your proteins?

2) Do you just not see any protein of interest by western, or do you just not see an overexpression (i.e. do you see the endogenous protein)?  If you see the endogenous protein (and the loading controls) then the western blot itself is not an issue.

Good luck!

Ariel

Hi,

There have been enough responses. Something that comes to mind is the timing of collecting your cells. It is possible that the proteins are transiently expressed and they have a very short half life. Also protein stability maybe an issue. So playing around with the collection time may give you better answers. I'd collect for both mRNA and protein and run them parallel at several time points. Not sure whether you have already tried this.

The first thing I would check is that your plasmid constructs are OK, they have a functioning promoter, there is a translation initiation consensus, etc. that your insert is what you want, no mutations, in particular with a stop codon close to initiation of translation.

then I would make a lot of controls for the western blot (endogenous genes, positive controls of transfections, etc) 

and finally I would check if the RNA is there (RTqPCR

if all this works there is still the possibility that these mRNAs are translationally controlled.

Best

Jose A.

We had a similar problem in our lab: there were no good antibodies against our protein of interest and despite high mRNA expression (qRT-PCR), we did not see the protein on our westerns (low sensitivity of antibodies). So we used a chimeric construct (protein+GFP) and detected the protein with an anti-GFP antibody on westerns. That worked! You could try this after having ruled out other western blot problems.

Hi ana.

It is noticeable that you have to check the all solutions and buffers that you made.check their pH and reliability. 

After that you have to check antibody the dilution protocol said.finally,try to understand your protein expressed in witch part of the cell.For instance,if it has intracellular expression you have to lysis your cells.

Best Regards.

Shahrooz.Ghaderi.

The western blot indicates that maybe the protein may not be expressed, even though you confirm DNA trasnfection was successful and confirmed via PCR. You may need to purify the target protein using its provided tag. Then you at least can make a conclusion that the cells are expressing the target protein.

You should always have multiple ways of detecting your tartget, so adding a tag is a wise precaution. I assume your protein is not a native HEK293T protein. Is your heterologous protein codon optimised for expression in HEK293T? If your gene has a deviation in its codon bias, this will result in low expression. In that case a synhetic ORF with added tags would solve your problem.

Thanks you all for your suggestions. I will try to answer to all your questions. 

the proteins in the gel should be at 60-70kDa. These proteins (SGLT1 and SGLT2) are endogenous proteins on HEK293/T cells (not sure about COS7) and I can see that on WB so the technique is not the issue. 

I have tried to observe the overexpression with a stable cell line but still no overexpression. the plasmid has a pCMV6-Neo vector but no tag. 

Sarah Kammerer how did you do the construct (GFP-protein)??? I do not have a tag like GFP because I am running NBDG uptake assays and the wavelength is the same and the tag would interefere on the results. 

All the buffers are ok because I prepared fresh to rule out that problem. 

My only suggestion is that the cells don't like the foreign DNA and for any reason they kick out the protein or the protein is silenced for any reason... 

Have you checked your expression construct itself? A simple thing to consider is a Kozak sequence at the beginning of the ORF. If it is targeted to an organelle, you will also need a targeting sequence.

Regarding your stable transfections the CMV promoter is sensitive to methylation.  Usually this is a gradual process but perhaps something that might be considered in this case.  Also, whilst you have verified the presence of the plasmid via PCR (a positive signal my not necessarily originate from DNA inside the cells) perhaps isolating the RNA and running a qPCR would confirm, or otherwise, whether or not you're actually getting RNA expression from the plasmid (ensure one primer is in the 3' or 5' UTR).  Maybe this would enable you to narrow your efforts.

Hi Ana,

if a gene fusion with a fluorescent protein is generally of interest to you and the excitation/emission sprectrum of GFP interferes with your other assays, then I'd like to note that there are several other colors available: http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/Articleimage/2010/IB/b926500g/b926500g-f12.gif

I don't know how your samples are treated between expression and application to the gel, therefore I have a treacherous question: Could it be that your protein is actually expressed, but you miss it because it is insoluble? If you lyse the cells and pellet the debris by centrifugation, an insoluble protein would also be pelleted and you would see nothing in the supernatant. I have had such issues when I tried to overexpress an E. coli gene in E. coli. If this appears to be a possible cause of your issues to you and you did not check that yet, just apply a piece of the pellet to the gel and you'll know.

Good luck!

Thank you John, Thanks an interesting question.

But if I apply the pellet it won'e mess up with the gel??

Thanks

Hi Ana, it can mess up with the gel if you apply too much. Just add a small portion of the pellet to the gel, maybe something in the range of 2-3 µl. Try to resuspend that in your SDS sample buffer, denature and ideally apply it to the gel while the sample is still hot (much easier to pipet then). You could also try to denature your entire pellet in something like 8M Urea or 6M GuaHCl, this should unfold and solubilize most any protein. I'd just play around a little and spend one gel for such a test. Note: As soon as a mixture of protein, GuaHCl and SDS sample buffer gets cold in your pipet tip, it will clog the tip; this really has to be pipetted while still hot. Hope this helps, even if it's just to exclude that your proteins are insoluble ;)

Thanks John I will try that. :) 

check whether

1 mRNA exists.

2 Your western blot system works well

3 Sometimes monoclonal antibody doesn't work. Polyclonal antibody is better.

I have the same problem actually and what i think is that the antibody is most likely the issue. This is common with proteins that are not highly expressed and which do not have specific monoclonal antibodies raised against them. I suppose the suggestion by sarah makes a lot of sense. Cheers.

Did you check if your protein can be secreted?...We had the same problem and in the end secretion was so quick that we found it in the medium and not within cells!!!