I have run a non-reducing gel with samples of a few antibodies. The samples are supposed to be pure, i.e. only containing the antibody of interest. I observe some bands below the main band. Does anyone have a suggestion to what this could be?
It is clear that the sample is not pure, additional lower bands at lower mass can be observed. The expected Antibodies bands are not quite overloaded. I may assume that some degradation occurs at amino acids level (e.g. deglycosilation which decreases the molecular mass).
It might suggest the presence of salt in your sample. Try using a desalting column (if you know for a fact that your sample has salt) and see. Since the bands is below, it might mean that you have protein degradation. The bands above your specific band might mean that there may be some type of post translational modification.
to Rasha Al-attar, PTM's usually increase the mass and it is more probably that PTM',s if present, are not distinguished so well in SDS. Mass spectrometry is the method of choice. Anette, I assume that your corresponding antibodies are the highest band in the gel, right ?
Thank you all for the suggestions. The band corresponding to the antibodies are the highest bands. To me degradation sounds to be the most plausible explanation. Sad but most likeley true.
I am though wondering what Rasha means with salt in the sample. Could a high salt concentration in the sample effect the migration of the antibody to this extent?
of ! the presence of salt will interfere with migration. Try acetone precipitation before running the gel. By using microcon columns for desalting you may loose some of your sample.
If you're talking about the faint bands just below the darker, highest bands of the gel, I have seen something similar with a protein I used to work with. Running a reducing gel removed the fainter faster-running protein band (or I suppose it could have removed the higher band), resulting in a single, tight band. Hence, I'm guessing, at least in my case, the two bands showed up due to different oxidation states. Good luck with your research!