I try to purify a protein (DEAE) but it stays with pigment and go out with wash (Pi 6.3 ; buffer pH 7.9): have you ever met this problem?
I work with a diatom
There are several reasons why a protein may not bind to an anion exchange column: (1) it doesn't have sufficient negative charge, (2) it is bound to something else that doesn't bind to the column (pigment?), (3) it is aggregated, (4) the ionic strength of the sample is too high, (5) the column is overloaded with protein and/or other negatively charged substances (such as nucleic acids), (6) the column was not prepared properly.
Hi Romain, it would help if you can clarify what pigment and what protein you are working with.
NADH and FAD dependent reducto/oxidases are known to bind dyes that have a similar structure as their cofactors.
Thank you for your answers. DEAE stock pH (after checking) was to 6.7, and buffer in the wash begining wich coming out of the column xas to about 8 so I think that the collumn was good. After I calculate the pI with protein calculator (it's sure if the protein is bound with another protein the pI could be change but past to 6.3 to more of 7.9 is really possible ?) after this protein is not a reducto/oxydase but it is possible that this protein is bound to pigment or membrane (surely chloroplastic). Thank you for your help, I think I will test a new DEAE with a higher pH and after I will see...
Thank you !
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