If you are over-expressing a gene, and no antibody is available, tag it with a his-tag (or your tag of choice) and use an antibody directed toward the tag to detect your protein of interest. Easy. 

If you are over-expressing a gene, and no antibody is available, tag it with a his-tag (or your tag of choice) and use an antibody directed toward the tag to detect your protein of interest. Easy. 

It would help to know what types of things you have tried to trouble shoot your WB first. If you have an Ab that has been used in WBs in another lab, you may want to start with the same procedure that that lab used (Similar extract, reducing/non-reducing gel, etc.).

From there I would seriously start questioning if my protein was being translated. Maybe try staining live or fixed cells on coverslips to see if the protein is there (this can be semi-quantitative with a calibratable light source) or  calculate MW and isoelectric point and try detecting it with silver stain on a 2D gel (with Mass spec. confirmation 2D gels and HPLC can be quantitative).

You could try cloning a epitope tag to your gene of interest and use a flag, myc, his, or HA antibody to detect it on blot. 

Lastly, if there are no good antibodies for your gene of interest, you could alway try cloning an epitope and either have an antibody made for it or send it out for antibody production. 

I hope some of this can give your some direction to go in.

Great suggestions from Luke and Justin!

Prior to giving up on the western, you need to determine whether your construct (CMV driven I presume) is working as it should (i.e., overexpressing your gene of interest). You can do this by using a bicistronic IRES vector. I would use some fluorescent protein (e.g., GFP) as your reporter so that you can visualize via fluorescence microscopy. Using an IRES containing reporter construct would also afford you the ability to avoid tagging your protein, which may impact the normal function of the protein.

You can also consider an ELISA. I have performed this using whole brain tissue lysates and it worked amazingly well, even though the assays are often geared towards measuring protein in supernatant or plasma. If I'm not mistaken, I think some companies are now selling kits that have been tested on whole cell lysates. Anyway, worth considering as ELISAs are more sensitive than WB. Also, antibodies that don't work by WB may work in ELISA since they may recognize the native protein better than the fully reduced protein by WB (can try a non-reducing gel as suggested already in WB).

Last resort would be to do an immunoprecipitation of your protein to concentrate your protein of interest, but having to do this may just indicate that your construct is not working as it should.

Maybe your gene of interest just needs to be subcloned into a different vector altogether.

Best of luck.

If you have access to a mass spec facility, there are many approaches to quantify the overexpression of your protein of interest.

However, as a direct answer to the question, Selected/Multiple Reaction Monitoring is the method of choice in absence of antibody for the protein provided you know what you are looking for.

Other alternatives then what people have mentioned would be-

Immunofluorescence staining (this is also useful to visualize the localization of your protein in a cell)

Stain your cells with antibody and FACS them (but this method usually needs a good permeabilization ). Both protocol could be found on abcam website.

Best wishes

Agree with all above and would add that you might consider a comprehensive  review of  the performance specs (information included with the product or call the vendor) for all the antibodies used - - - some just don't work well in Western Blots, perhaps due to the fact that they recognize linear epitopes (denatured) vs. conformational (native).  Also review the nature of the immunogen (e.g. peptide or protein).  If the protein was used as an immunogen, review if the expression system was prokaryotic ( i.e. E.coli) vs. Eukaryotic (CHO) or insect ( sf-9).  Sometimes, post-translational modifications make a difference and these expressions systems will vary on that.

Time to time I have proteins that are highly regulated. I can have a great mRNA level by my over-expressing system, but no protein at all (translation blocked by miRNA or lncRNA...?). I should advice as peter to do a mass spec to be sure that your protein is there, and to put some positive controls in your Western blot to be sure that it is an antibody issue, rather than a toxic or extremely regulated protein in your specific cell line.

Hello

A functional assay specific for your protein of interest can be performed instead of western blots. That is what many labs working on membrane proteins do, as it is hard to find antibodies for these proteins.

Good Luck.

An easy start for troubleshooting is to start with a positive control.  Companies that sell antibodies will often sell the purified protein as well.  Or (and more realistically), see if you can identify a cell line or tissue that DOES express the protein endogenously.  Then do your Western blots with that and use that for trouble shooting.  If the positive control blots and the cells you've transfected don't, it's probably an issue of poor expression (plasmid problems, transfection efficiency, insufficient level of expression to be detectable, failure to express for other reasons).  If the positive control also doesn't blot, then it's your technique or the antibody and if 5 different antibodies that are said to blot don't work then perhaps your technique. You can also contact technical support for the companies that sold you the antibodies as they can advise you about how much protein to load and what conditions to use.

Tagging the protein is a good option but sometimes is the quantity of the protein in your cells that is the issue although you are overexpressing the protein, then it should not be an issue. I would try to overexpress the protein in a cell line  that is good in overexpressing protein such as 293T and if you do not see any results from the antibodies in WB I would move to the tag-protein

you can transfected the your tumor cells GFP tags gene of interest , do confocal to check the expression intensity and compare with other normal transfected cell.

Or try epitope labeling for example if your protein of interest have more than one sub-units try to use antibody for one its sub-unit specific.

What gene?  I've run into this problem with Wnt proteins before. The bands on my blots are hopelessly weak, despite qRT-PCR results indicating expression on par with actin. In my case, this is because the proteins are immediately secreted. There are two ways to solve this problem if your protein is secreted:

You can run the medium (requires immunodepletion and concentration - can be expensive). Alternatively, you may be able to find a pharmacological inhibitor that targets the secretory pathway for your protein. This will cause it to build up in the cells, and will give a much better band on the western. 

If your protein is not secreted, I would GFP or poly-his tag it, as previous posters have suggested. 

Most antibody vendors provide some sort of guarantee. See if you can work with them to get a functioning antibody. 

@all- thanks for sharing your experiences and great advice/suggestions

@Carl Manner- indeed is the Wnt pathway relation protein but receptor: Frizzled receptors 9 and 10...any suggestion now?

I have a stupid question concerning your western: are you using RIPA buffer to do your lysis, or a high salt, SDS hot, or directly laemmli buffer? Receptors are in the membrane and some buffers are not strong enough to lyse the cells (RIPA is more for cytoplasmic proteins and when you centrifuge your sample, you loose the DNA but some membrane as well as some receptors/transcriptor factor). It happens to me with HER2, I compared RIPA with leammli and the results were drastically differents...You can do your own laemmli buffer very easily without blue and quantify with some kit SDS friendly (as Biorad DC protein assay).

I would also try several things 1. Use a positive control for your protein of interest. 2. Transfect an easy cell line e.g Hek 293 3. If your want to enrich use a cell fractionation approach like this one

http://cshprotocols.cshlp.org/content/2009/12/pdb.prot5330.full

I have to say that functional assays would be nice but I guess without showing an increase of your protein by western blot it will be very had regarding argumentation

Regards

Sven

@ Fazel,

this will only work if your transgene expression is pretty strong or if you would do an IP which again needs an working antibody. For a moderate overexpression I doubt your would see it.

This is right it is a easy and quick method. I have to say that pcDNA vectors in my hands were quite difficult especially because I work with hematopoetic cells and derivatives and CMV promoter is not optimal for this cell systems. My only concern was that low expression will not result in additional bands after Coomassie staining

Regards

Sven

You may develop an SRM assay for your protein of interest and quantify it through mass spectrometry in your experimental conditions.Absolutely no need to look for antibodies.