Hi everyone,

I'm working with a protein of interest (POI) that is approximately 200 kDa, which I'm producing in E. coli. After cell lysis and Strep-tag purification, I can isolate my protein, but I still have some impurities. Based on SDS-PAGE analysis, my POI is by far the largest protein in the sample, with the next largest impurity migrating between 100 kDa and 70 kDa under reducing conditions.

I know that Superdex 200, when used on an ÄKTA system, should effectively separate my POI, as it would likely elute close to the void volume, while the impurities should elute later. However, I don’t have easy access to an ÄKTA system at the moment. I’m considering using gravity-flow size exclusion chromatography (SEC) instead, but I’m unsure about the column height required to achieve sufficient resolution.

Has anyone had experience with gravity-flow SEC for proteins of this size range?

Specifically, I’d appreciate any advice on the appropriate column height, resin choice, and any other tips for optimizing this approach.

Thanks in advance for your input!

Best regards, Niklas Eckert Elfving