Hello everyone,
I am doing gel filtration chromatography with a protein I purified from Ni++ IMAC. The protein however seems to be coming out before the expected elution volume.
However, when I run the eluate on a PAGE gel it ties with the molecular weight. I am curious about the disparity in these two results.
I will be grateful to hear from you.
Thanks
Earlier elution from a size exclusion column than would be expected for the monomer indicates either that the protein has a non-globular shape, or that it is in an oligomeric quaternarry structure, or that it is aggregated. In each case, the protein will run on SDS-PAGE as the monomer. If you have access to a dynamic light scattering instrument or SEC-MALS setup, you can use it to check the molecular weight of the protein in the size exclusion column fraction.
As Dr Shapiro said above, protein eluting earlier than the expected elution volume would indicate oligomerisation (an ordered process) or aggregation (a random process), or non-globular shape of the protein. This happens very often. If your protein elutes around the void volume mark (i.e. at the point where anything that is totally excluded from the column pores would elute - check your column manual or calibration), then it most likely that this is an aggregate, and that is very bad news.
If by "low volume range" you mean that it's eluting later than you'd expect, then on the other hand this could mean that the protein is "trapped" in the column for a little while - it interacts with the resin. This can happen if the ionic strength of the buffer is too low. E.g. I keep at least 300 mM NaCl in all my GF buffers.
In either case, if you want to rescue your protein, you should test a few other buffers, changing pH as well as increasing ionic strength. Depending on what you want to do with the protein after, adding a mild detergent might also help.
I agree with the comments given above. In addition, you need to consider parameters, such as flow rate, fluctuation of system pressure, etc.
Just to add further, proteins from the class, Intrinsically unstructured proteins" are loosely packed in a 3D structure, and therefore these proteins have relatively higher hydrodynamic radius as compared to globular proteins of a similar molecular weight. In such situation, this is neither a case of oligomerization nor aggregation, and the protein will still elute earlier than expected. You may learn more about your protein from computational tools, and you may get an idea about the shape and structure of your protein.
Everything was said before, first I would run the size exclusion chromatography with a protein of known size to be sure the column setup is ok. Than I would suggest you run a native PAGE instead of SDS-PAGE, it will tell you more about how the protein behaves in the column.
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