debmukh at scripps.edu (Debashis from San Diego) wrote:
>>We have been trying to purify (and subsequently crystallize) a cytosolic
>kinase from a hyperthermophile, the gene being cloned and overexpressed in
>E.coli. Affi-Gel Blue was found to be most suitable for chromatography and
>everything looks great upto the sample loading stage. Then the panic
>starts as the protein never comes out even with 3M KCl in the eluting
>buffer. It seems something is wrong at this high salt conc. I was thinking
>of using NAD+ as elutant.
>>Any idea, help, suggestion, reference (as I'm a novice in the field) would
>be highly appreciated.
Dye chromatography is a very powerful purification... when it works.
In too many cases it is plagued by very low recovery of the protein.
One thing to remember is that the binding mode is complex mix of
ionic and hydrophobic interactions. If KCl at 2 M does not elute, it's
time to try something else. 50% ethylene glycol with 0.5-1 M salt,
dialysable detergents, Mg+NAD, Mg+ATP in the elution buffer are
things to try.
DK
