I have purified my HIS-tagged protein with 1% Triton X-100 in the wash and elution buffers with a Nickel affinity column. The protein was over-expressed in E. coli and as it's a membrane protein, it's quite hydrophobic.
I want to purify my protein further by use of Gel filtration/Size exclusion chromatography if possible. I'm just afraid that if I don't use Triton X-100 in the SE buffers, that my protein will precipitate out onto the column. I do want to remove most, if not all, of the Triton X-100 in the end.
Will the SE column only remove the Triton that's not associated with my protein or will it remove all of the Triton and thus cause my protein to precipitate?
Hi there,
If the SEC buffer doesn't contain any Triton, the protein would eventually precipitate because of Triton dilution. If you want to lower Triton concentration and test protein solubility, it's easier to do it when protein is adsorbed on NiNTA column by washing away excess of Triton with a buffer containing Triton at it's CMC. If working at concentration lower than CMC you will tend to dissociate Triton molecules from protein containing micelles.
Thank you Dominique! I'll give that a try. Is there a chance that the protein will still precipitate if I wash too long with the Triton at its CMC, or should it be relatively safe?
It should be OK as Triton molecules are in equilibrium between monomeric and micellar forms. If working at CMC, any further Triton molecules will try to form micelles. If washing micelles at CMC there shouldn't be any dissociation from micelles.
I agree with Dominique; however please keep in mind that cmc of any detergent slightly depends on the ionic strength of the buffer you use. CMC in 50 mM phosphate buffer, pH 7 is not equal with cmc in 50 mM MOPS buffer, pH 7. Thus it is always "safe" to use detergent concentration in all buffers a little bit above the "well-known" and "published" cmc after solubilization.
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