I performed affinity pull down using peptides and subsequently performed Mass Spec analysis. A protein that showed significant interaction with the peptides in my Mass spec analysis does not show up on the western blot on detection with monoclonal antibodies against that specific protein, on performing the exact same affinity purification. Apart from Mass spec being a rather sensitive technique, could there be any other plausible reasons for this incongruent observation ?
Hi,
Recognition of your protein of interest by a monoclonal antibody might be dependent on structural properties in the protein of interest that are lost upon gel electrophoresis and transfer.
Did you try if you could pull down the protein of interest using the monoclonal antibody instead of the peptide(s) you've used before?
BTW, how are you detecting any potential proteins recognized by the monoclonal antibody; a secondary antibody?
Hope this helps!
Hi Arsh,
As you have already mentioned the two methods have total different spectrum of sensitivity for detecting the target protein/peptide. There are several factors that can bring difficulty in identifying the proteins/peptide by conventional WB like, pI of your target protein or the epitope to which your MAb is binding is well exposed or not and so on.
I believe you must have tried all these preliminary ideas to identify your protein through WB. Since you mentioned that you are using affinity purification to fractionate out your protein of interest, I believe you must have a tag added to your protein. You can probe your blot for Ab against the tag you have added to your protein!
Best,
So this is what my concern is currently. I have tried two different clones of monoclonal antibodies for WB analysis: directed against the N-terminus of the protein of interest. When I go back to my mass spec data, I see it is a short stretch of peptide in the C-terminus of the protein that got detected, so now I intend to try a different antibody with its epitope in this specific region. For the AP I am using biotinylated peptides as the bait to pull down interaction partners from cell lysates via streptavidin-sepharose affinity resin. I also tested for the positive controls for this interaction on my WB and could confirm that the pull down worked fine.
I don't think that there is a relationship between the epitope(s) exposed for either affinity pull-down or WB analysis on the one hand and stretches of amino acid sequences identified in an MS-based analysis of the affinity-purified proteins, so I doubt whether a mAb directed against the C-terminus of the protein will yield significantly better results.
During affinity pull-down (either using biotinylated peptides or a mAb), the analyte proteins are in 'native' condition and therefore able to bind the scaffold.
Once you've recovered your proteins of interest and put these on a gel for WB analysis, protein structure -and therewith the epitopes- dissappears, making it harder to specifically detected your protein(s) of interest in such a setup.
There is no relationship between the ability to detect a protein by western blot and mass spectrometry. There are hundreds of proteins that cannot be detected by western blot that are rather easy to detect by mass spectrometry, especially when using monoclonals. Polyclonal antibodies are usually much, much better for western blots than monoclonals, so if you are not being successful with monoclonals, then either use an epitope tag, go for a polyclonal, or stick with mass spectrometry, which is the gold standard for detection of specific proteins.
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