tob at ssi.dk
tob at ssi.dk
Fri Oct 22 03:15:18 EST 1999
I believe I used the wrong expression in my latest answer on this post. Instead of calling gel chromatography the best way to look at aggregates, I should have said the easist way, as obviously Keri already has the equipment to run chromatography. For analytical ultracentrifugation you need equipment and expertise to set up the method. As Keri obviously can tell, by size exclusion chromatography, that she has an aggregate I believe this low resolution technique is good enough for her application. I agree that light scattering could be an obvious choice, but Matt how do you detect an aggregate like protease-inhibitor or coil-coil or cysteine bridge aggregates by light scattering? I believe the terminology here is a bit confusing. We need to separate: the aggregates that are created by recombinant protein expression and purification methods, and the complexes that are created by "real" affinity between proteins.
At last to Keri. A beneficial side-effect of using size exclusion chromatography is that you will get a pure monomeric preparation that you could use for NMR structure studies. I guess that this is the goal of your study. I want to warn you that some proteins have a strange behavior in size exclusion chromatography. Rod-shaped proteins have a larger Stokes radius than the MW indicates and therefore seems much larger than their actual MW on size exclusion chomatography. Different additives like detergents can also make your protein look larger, due to incorporation in detergent micelles.
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