My protein of interest gets detected in IF, but not by western. Have used different tags. GFP and HA. Both works fine in IF. Protein is expressed and localized properly. No cell death or protein aggregation/degradation issues, based on IF info. Absolutely no signal in untransfected cells. Transfection efficiency was fairly good- 50-60%.
But only the GFP fusion gets detected in western, but not the HA-tag fusion. Absolutely blank lanes, no sign of degradation products/smear either. The same insert got cloned into both vectors. Both were N' fusions and were sequenced to confirm their fusion and frame. No question of epitope masking issue as it is an SDS-PAGE-Western.
IF on fixed tissues are a general problem with many antibodies that are good in westerns. An Ab that is good in IF, mostly works fine with open epitopes found in westerns. But this reverse and unusual problem we have is maddening and will be glad if anyone has any thoughts/suggestions.
If your IF antibody recognizes a conformational or discontinuous epitope, then it would not be surprising that it doesn't work in a Western blot format. Do you have any detailed information about the epitope that's recognized by this antibody? If it is a commercial antibody the company should have some information about it.
Alternatively, your protein might adopt some non-natural conformation on the Western blot membrane, which is rare but not unheard off. You could try to add another denaturation step: after the transfer treat the membrane for 5 min in 1 N NaOH, then wash, block, and treat as usual. It might remove any non-natural conformation and render the epitope accessible. It has worked for me more than once.
If your IF antibody recognizes a conformational or discontinuous epitope, then it would not be surprising that it doesn't work in a Western blot format. Do you have any detailed information about the epitope that's recognized by this antibody? If it is a commercial antibody the company should have some information about it.
Alternatively, your protein might adopt some non-natural conformation on the Western blot membrane, which is rare but not unheard off. You could try to add another denaturation step: after the transfer treat the membrane for 5 min in 1 N NaOH, then wash, block, and treat as usual. It might remove any non-natural conformation and render the epitope accessible. It has worked for me more than once.
Thanks Holger. Yes, thinking back, it just occurred to me that it is possible that the monoclonal antibody may be recognizing a complex 3D epitope, which is intact in fixed cell samples but may have got disturbed in westerns and therefore difficult to detect. But the Ab data sheet shows clear westerns with untagged proteins. Possibly, the GFP being a bigger tag, may have partially helped to retain the epitope, while the small HA tagged is not able to do so. The highly charged small 6X His-tag is known to cause problems in protein folding, but not sure about the HA-tag. Now I am thinking of using a polyclonal antibody for the same protein and see if it helps. Will also try your suggestion of 5 min incubation with 1 N NaOH post transfer. I hope we could resolve the problem, if everything else about the constructs are fine, as we believe. Can't wait to check it ASAP. Will post my observation soon. Thanks again.
Upsetting that the product data sheet shows a good western, but its just not working for your Western. There are many of processing/condition differences that could cause that so if you can't determine the reason pretty quickly, its best not to get bogged down trying to figure out why.
It seems you have good handle on what you are doing with westerns, and Holger has given a good suggestion above.
I'm not quite clear. Are you using anti-HA or a protein specific antibody? If you are using a protein specific antibody, might it be useful to use an anti-HA antibody for the western, thus avoiding problems with possible conformational differences between IF and Western?
As you state above, a polyclonal gives you more chances for success for protein specific antibodies so that is certainly a way to go too.
Another possibility, if a LiCor Odyssey (or similar) system available to you might be their "In-Cell Western". https://www.licor.com/bio/applications/in-cell_western_assay/?gclid=CPG_g9rq_80CFZA2aQod5OkAPg
There is a significant start up cost if you don't already have the materials for this the LiCor Fluorescent western detection, but that is what I thought of when I read your initial question: "Is it possible that one can detect a protein by immunocytochemistry but not in western blotting? "
https://www.licor.com/bio/applications/in-cell_western_assay/?gclid=CPG_g9rq_80CFZA2aQod5OkAPg
Thanks Floyd. An yet to see the results with polyclonal antibody. The ab is against the protein and not the HA tag. But same problem persists with HA tag ab as well. Weird. GFP fusions are fine. We have licor ab too. Will checkout.
@ Indumathi- I am having the same problem of detecting my protein using anti-HA antibody by western. My protein is a 3XHA N-terminal fusion. I am curious to know if the NaOH treatment worked in your case or not
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