I used chromatography to purify my protein and now I have fractions with different concentrations ranging from 1-16 mg/ml. Should I pool all these fractions or just the ones with high concentration (where under the actual peak)?
Thanks in advice
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1$\begingroup$ How pure do you need your product to be? $\endgroup$– WaylanderAug 8, 2019 at 7:53
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$\begingroup$ I never purified proteins. In column chromatography all fractions containing "just" the target compound(s) are mixed together. $\endgroup$– AlchimistaAug 8, 2019 at 8:56
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1$\begingroup$ You should run gels on the fractions and verify purity, discuss with others in the lab including your supervisor, and then pool as necessary. Also, keep the fractions at the recommended temperature (probably in a cold room) until then. $\endgroup$– Buck Thorn ♦Aug 8, 2019 at 9:55
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If all fractions contain the same protein, I'd combined all and concentrated the combined solutions to desired concentration using biochemical techniques such as dialysis. If they contained more than one protein, then I'd combined all fraction collected under the correct peak. However, it is better ask your supervisor (or adviser) before you combined them. The person who wrote the protocol knows which one to collect. Yet, you can look at following reference to get an idea how to concentrate the combined protein solutions:
Sarah M. Andrew, Julie A. Titus, Louis Zumstein, "Dialysis and Concentration of Protein Solutions," Current Protocols in Immunology 1997, 21(1), A.3H.1-A.3H.5 (https://doi.org/10.1002/0471142735.ima03hs21).
answered Aug 8, 2019 at 21:57