A lab colleague made an antibody specific for our protein of interest. The antibody shows many bands via western blot, and when I go back and use the commercially available antibodies it also detects more bands than what the company reported. I did a PTM prediction using my sequence and I have many PTM predicted events. What is the best way to determine the PTM? I have used a recombinant protein (has less bands but some corresponding to the bands observed in different cell types) knockdown and overexpression plasmids and all of the bands change depending on treatment (there is not one specific band down or up dependent on knockdown or overexpression).
"What is the best way to determine the PTM?"
I would suggest PTM analysis by mass spectrometry, in my opinnion the easiest and accurate way of PTM determination
If mass spectrometry is not available, you could try staining for carbohydrates. Glycosylation will increase the apparent Mw of a protein on SDS-PAGE, not only because it adds extra atoms to the molecule, but also because it reduces binding of SDS. If the extra bands have a lower Mw than expcted, the most likely explanation is proteolytic degradation. Finally you should also consider the possibility of proteins of similar sequence cross-reacting with your antibody.
The best way would be Mass Spec as mentioned above.
I got a bit suspicious that your "specific" antibody seems to show multiple bands in your western blots, even for a recombinant protein. Is your recombinant protein also post-translationally modified or unstable and shows degradation bands? Otherwise you would expect a single band in your blot, wouldn't you? Is it a polyclonal or monoclonal antibody and do you know how it was generated and purified? E.g. if someone used e.g. a GST-fusion protein you may have GST-specific antibodies in your antibody mix which could give you additional signals (this would not be seen when you checked the antibody with recombinant protein) unkless you affinity purify the antibody with your target protein or remove GST-specific antibodies using GST on beads. Another possibility is also when peptides were used to generate the antibody which could lead to antibodies reognizing similar peptides in other proteins in a lysate, etc. So, to cut a long story short, make sure that you are working with a really specific antibody.
I did speak with our proteomics center because I think Mass spec is the best way as well and he said that he could easily determine the proteolytic cleavage sites but I would need to run mass spect for each band to verify if it is in fact my protein. He said to check glycosylation and phosphorylation via MS is also a bit more difficult. Also, with MS the actual PTM or proteolytic cleavage site could not be determined. It would just tell me if SPA is there or not. I also have looked at trying to identify the glycosylated proteins by staining the membrane, but my worry is that how could I determine that what is glycosylated is actually my protein of interest and not some other protein? I have been looking at Proximal Ligation Assay but this has its caveats as well.
As I did state above I have the same bands using the commercially available polyclonal antibody. So I do not think it is the way the antibody was generated. The mouse antibody generated in our lab was also polyclonal.
Skylar, some general aspects that will help explaining your observations : How pure is the antibody you are using, how has it been generated and how has it been purified? Is it against the whole protein (purified from a lysate or is it a recombinant tagged protein?) or has it been raised against a short synthetic peptide?
It also could help to use a monoclonal or an AB that has been generated against a short defined synthetic peptide in order to reduce the amount of bands, but you might miss valuable data about PTMs and processing. So maybe just follow several bands until you have more insights what's actually happening.
Otherwise, you could check the protein sequence of your protein to explain potential cross reactivity of the antibody with other proteins. As you are seeing some bands also when overexpressing your protein, they could be the result of PTMs (e.g. tyrosine phosphorylation may shift a few kD), alternative splicing or degradation. Isolating the bands (preferably after 2D electrophoresis) and identifying them by LC/MS might give you valuable insights.
Dear Skylar King
you is a complex question and much more detail are necessary to provide a reliable answer.
what do you mean for multiple bands? Which is the relative intensity of those bands? one principall at the right MW and many minor or some comparable? And those bands were detected by performing a WB by loading the recombinant protein and using the antibody as primary antibody in WB or loading cell lisate of cell that contain an overexpressed form of your protein of interest?
And the antibody made from your colleagues is monoclonal or polyclonal?
Because for example if you are using a polyclonal antibody is it possible that in case that it was produced by immunizing a low purity protein preparation , the antibody mixture may contain also some antibody specific for the impurities and you will se a multiple band in a WB expecially if you are performing it in a cell extract.
On the contrary if the antibody is a monoclonal antibody able to recognize linear epitope and the WB is directed against the purified protein of interest, the precence of multiple bands, is more probable that the other bands are fragments of this protein that contain the same epitope.
Ciao
Manuele
Specific protein PTMs can be detected by mass spectrometry. Mass spectrometry can distinguish the molecular weight changes of proteins before and after modification. Therefore, as long as the molecular weight changes of the target protein are known, the type of PTMs can be identified and quantified.
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