A chromophorylated protein is dissolved from a washed pellet fraction by 2% CHAPS, which preserved the color of the protein. Anything against subjecting this soluble prep to SEC in the presence of CHAPS? Which concentrations of CHAPS are safe in this case?
To keep the protein from precipitating, equilibrate and elute the column with a CHAPS concentration that is at least a few fold above the critical micellar concentration of CHAPS, which is 6-10 mM. The higher the protein concentration, the higher the CHAPS concentration should be. You want there to be enough CHAPS that each micelle of 10 CHAPS molecules contains no more than one protein molecule. Because CHAPS micelles are pretty small, having the protein within micelles should not seriously affect its separation from much larger or much smaller molecules, but may reduce the ability to separate it from similarly sized proteins.
Also be aware, once you have exposed a gel-filtration resin to detergents, you will not be able to completely remove it from the resin anymore.
You will find many recipes for washing the column with all kind of solutions to remove the detergent, but in my experience, there is always a residual concentration that remains behind and changes its behavior.
So, plan ahead and only use a column you can spare or dedicate to this purpose.
You can start trying with 2-3 fold the CMC of CHAPS (8 mM, so keeping 1% (16 mM) could be a good start). CHAPS micelles are pretty small though, so even having some excess must be good for you. Please mind that, depending on your downstream application, you may want to keep your detergent concentration as low as possible (structural studies), so maybe you will have to try several concentrations of CHAPS in buffers.
After solubilisation, proteins are in mixed detergent-lipid-protein micelles, the solubilisation protocol has to ensure that each such micelle contains at most 1 protein molecule (otherwise, further purification will not be possible). At the same time, lipid/detergent ratio needs to be so high, that annular lipids are not stripped from the protein, usually this requires addition of lipid as well as detergent.
Once you have solubilised the protein, you need to maintain [detergent] at the cmc. Higher [det] will strip annular lipids and inactivate the protein, lower [det] and detergent will leave the micelles, leading to precipitation of the protein, which is often not soluble by itself. The cmc depends on temperature, [salt] and other factors, you have to determine it for each buffer separately.
Each SEC column needs to be equilibrated with the detergent, this can take several days of passing buffer over it in a closed loop. Achim Recktenwald added a valuable point: a column that was in contact with detergent should be used only for chromatography with that particular detergent.
Engelbert Buxbaum: Would it perhaps make sense for him to sonicate the protein / CHAPS solution, to make the micelles small and increase the chance of having only 1 protein / micelle?
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