Hello,
So I recently expressed a protein of about 25 kDa. I first purified it through Ni-NTA affinity column, then ran O/N dialysis, then for a final stage of purification (because I want to crystallise it) I ran it through a sephadex G200 resin. However, it resulted in the attached chromatogram: 3 peaks!
I tested each of these and they all show activity for the protein so I am sure that they are all related to the protein, and SDS PAGE only shows one band for each of the peaks, corresponding to my protein.
Has anyone experienced this before? Please take a look at the attached image.
Hi there,
Your protein forms multimers. Your 3 peaks correspond to three major states of oligomerization (monomer/dimer/tetramer for instance). If you want to cristallize you have to purify one specie first. To do so I would suggest you to set condition (mainly pH and ionic strength) where one of the specie is vastly favored toward the others.
Thank you for your quick response Dominique. I had considered this, however, wouldn't dimerization/trimerization result in peaks that are far more separated considering that the MW would basically double/triple?
Hi again,
Column calibration will actually tell you but gel filtration is definitely not very resolutive.
I agree with the Dominique's view. While you run your protein on SDS-PAGE you are basically unfolding it and so a single band is expected but you may observe different band patterns under a native gel.
Sometimes you can have slightly misfolded protein that runs as a second peak. For example I have a protein that gives 2 peaks in a Superdex75, with one peak that corresponds to the correct size and the other one slightly larger but not enough to be a dimer. Both present activity and binding to ligands and both give similar sizes in DLS, although the misfolded gives just slightly larger. My guess is that there's a population of slightly misfolded protein.
Altough Dominique is right, with a G200 you have to be sure with calibration.
Another way to know if those peaks are dimers or multimers is to run again each peak separatedly. If they are dimers/multimers, they should form again, and you should see 3 peaks. If you see 1 peak, it's misfolded protein
Run blue native page to see if it is misfolded protein or dimers/multimers
Hi Bryan,
I would suggest that you run a Native PAGE (either blue or clear) as suggested by Omar above to have an idea on the oligomers that you have.
I would suggest also to screen different additives (EGTA, Calcium, Magnesium, NAPDH..... of course according to your protein domains) this can be also done using a Native PAGE.
as last resort you can try to make very small fractions during your SEC run to limit the cross contamination of your different oligomers and of course crystallize the 3 fractions separately.
Good luck
- Badges
- Science method