Dealing with aggregate and high protein concentartion

[hplcchat]

Is there a SEC metod to analyze the aggregate level for high concentrated proteins? Does anyone have an experience using a low volume injector (25uL)? protein concentartion that i am working with is about 100mg/mL. I am avoiding dilutions and making neat injections of the sample since changes in concentartion will affect the %aggregate.

[danko]

If the aggregates are not stable (which is your assumption) I doubt you can do very reliable quantitative determinations.
There is quite considerable dilution going on under the chromatography itself, which in turn, will facilitate the disintegration of unstable aggregates.

Best Regards

[biochemist]

Hplcchat,

Have you tried using native PAGE with densitometry? In our experience, it can detect lower levels of aggregates compared to SEC.

Out of curiosity, is the protein concentration really that high? If the formulation of the protein requires dilution, then the formation of reversible aggregates shouldn't matter, would be my guess.