My protein is quite a large one and in theory should be easily separated by SEC. It does and I get quite pure fractions. The thing is my protein elutes in several different peaks i.e sizes. I changed the salt content of my buffers and the same thing happens. Also DLS indicates that the samples are mono-dispersed. An SDS gel suggests that all peaks belong to my enzyme with very little contamination from other proteins.
Your protein may interact with the size-exclusion column, causing it to elute broadly. A different resin chemistry may help if this is the problem.
Are you working with a DNA binding protein? and is the column matrix made of sugars? Then it is likely that your protein is binding to the column matrix.
Hey guys several distinct clear peaks are formed. Also one of the peaks seems to elute with one of its substrates (NADH)
Hello Karthik. Thanks for your answer. No my protein doesn't bind DNA.
Then they are likely to be different functional/oligomeric/structural states. SEC-MALS or AUC might give more information than DLS. Sounds interesting. Good luck.
Within each peak there is mono-dispersion by DLS but if peak 1 sample was mixed with a peak 4 sample would there still be mono-dispersed behavior (was the load solution mono-dispersed)? The molecular weight standards ran well I assume since you have sharp peaks. The SDS-PAGE of each peak showed the same mass reduced and non-reduced, right? What is the backbone chemistry of the SEC? If there's no column interaction and this is a relatively large mass, what ever that might be, a single polypeptide chain or subunit system might exhibit different conformational states or Stoke's radii that affect migration on SEC. Subunit A or domain A might be differently shaped than subunit B or domain B and so forth so that the various combinations give rise to different morphologies (peaks) not discerned by DLS. Is the specific activity identical in each peak fraction of each peak? If there is a genuine mass difference then can you do MALDI on each peak? Is it possible that the there's an equilibrium between various forms and running SEC shifts the equilibrium on the column but then the equilibrium re-establishes in the pool of each peak? So if you re-inject a peak would you get the same 4 peak distribution?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins