I agree with Abdelhalim, proteolyic degradation of larger, very abundant proteins has 'contaminated' lower regions of gels I have submitted for mass spec. Make every effort to use fresh lysates and use protease inhibitors if possible.

Hi. If the high MW protein is very acidic then it can run faster than expected on SDS PAGE. You could try native (non SDS) PAGE. Or use SDS PAGE using a different buffers than tris-glycine.

Hi Kalina,

I am not sure to understand well your issue. It is true that a number of proteins have anomalous SDS-PAGE migration for different reasons but it is not normally a problem that one should worry about.

I am deducing that you are cutting the bands between 10-15 Kd and sending them to Mass Spectrometry for protein identification. In this case if you have proteolytic products mass spectrometry cannot tell you that. You have to deduce it yourself. Remember in mass spectrometry you will have to digest your protein mixture to smaller fragments. So, how can you be sure you don't have proteolysis either during the extraction process or during the purification if you perform any?

Also a nice band on coomassie would probably correspond to several different proteins.

I agree with Abdelhalim, proteolyic degradation of larger, very abundant proteins has 'contaminated' lower regions of gels I have submitted for mass spec. Make every effort to use fresh lysates and use protease inhibitors if possible.

Thank you very much!

I will try to use protease inhibitors on next collection!

I don't purify my samples but I do concentrate them overnight at 4C against polyethylene glycol in 3kDa-cut off membranes. The western blot was done on the following morning after the concentrated pellets were reconstituted in ice cold PBS and kept on ice at all times after that.

Thank you all!

why dont u try doing pulse field gel electrophoresis? it has electrodes arrangd in hexagonal around the gel so it might be helpful in separating the bands effectively. i've known this technique to be used for DNA analysis. but i think same concept can also be used for protein

I agree with Matt. Your protein must have degraded during o/n concentration. Try to reduce this time and do use glycerol (atleast 10%) + protease inhibitors + pmsf to prevent this degradation. save ur protein by aliquoting them in small volumes (one loading at a time).

Yes your protein must have degradated either during concentration or purification. Always is is better to do the preparations in a shorter time span. And during the lysis preparation it is advised to use protease inhibitors.

It would be good if you can include a positive control for your antibody. This will help you troubleshoot. Perhaps this is not so easy, since you are trying to purify/identify your protein, but at some level you know the antibody works, whether in immunoprecipitation or Western blots. Perhaps you can immunoprecipitate then run that on a gel for Western as a control. So if you can use it during your purification process and Western it will give you information about what is happening.

Do you run some lysate before any treatment to make sure that your protein of interest was there to begin with?

It's also possible that your protein of interest is being lost during the concentration process - it might be stuck on the membrane after you resuspend in PBS. If you need to concentrate your sample, you can try precipitating all of the proteins in TCA and then resuspending that pellet in boiling sample buffer - this ensures you don't lose anything. Feel free to message me for a more detailed protocol.

If you're having trouble separating proteins or protein fragments of similar size, you can try a gradient gel (for example, 4-20%).

Are you shure that your western blot worked? Like you have written that on CBB stained gels you have seen a corresponding band but it was absent in the western blot.

However, that MS analysis revealed high MW proteins can happen, but were the results significant? Otherwise they can be false-positives.

Thank you! - I had a positive control for the antibody - it worked well. I will probably try immunoprecipitation also as a next step! Thank you for all suggestions!

When doing western blots, nothing is perfect, and your protein of interest can give rise to higher molecular weight bands (multimers, homo and hetero) as well as proteolytic degradation products of lower molecular weight. Any protein can degrade before, during, and after extraction, even during the boiling with reducing/denaturing agents some peptide bonds can be more prone to hydrolysis than others, giving rise to specific lower molecular weight proteolytic degradation products. Degradation of proteins can also happen before you extract, so sometimres even with protease inhibitors, using ice cold solutions and working on ice doesn't stop getting smaller bands. Multimers can form via disulfide bridges that emerge in the stacking gel where all proteins form a sharp concentrated front moving towards the separating gel, but the DTT or beta mercapto ethanol from the sample buffer will stay behind in the well. You can add Iodoacetamide (10 time excess compared to reducing agent) to block the free SH groups after reducing denaturation, and that will certainly reduce the amount of higher molecular weight bands, but few people seem to know about this. You need positive controls (ideally a pure protein) and negative controls on your gel so that you know what's going on. Negative controls are not always available (like a mutant in which the gene encoding your protein is knocked out), but if you express a recombinant protein in E.coli. the control is obviously the untransformed E.coli. You cannot do too many controls.

I agree with Abdelhalim and Matt, you probably have proteolysis in your sample, but, did you find, by MS, all the proteins of the band below 50KDa?

You should boil the sample prior to loading

To decrease the free energy of the system

Are the peptides that were measured proteotypic for the protein you are looking for or could they stem from other proteins as well? This would answer whether you are truly looking at the protein of interest. If none of the peptides are proteotypic, try to search for a known protetypic peptide for your protein using SRM or MRM.

Do you have any sequence (MS/MS) information or was the assignment done by PMF? In case MS/MS wasn't done make sure to do it to confirm that the resulting sequences actually confirm the finding.

What is the sequence coverage and how are the matching peptides distributed over the sequence? This would give you an idea of which part of the protein you are looking at. If the peptides are from just one area of the sequence than you are on to a fragment of the protein (keep in mind proteotypic peptides must be there to be able to draw any conclusion unless you can rule out the existence of other proteins by different means).

I suggest you answer these questions before you tweak your experimental settings.