AEVDOKIMOZ at cinci.rr.com
Fri Sep 28 22:06:46 EST 2001
One more thing to consider is to screen wider array of resins - if memory
serves, there are green, blue, red, yellow and polka-dot-aquamarine resins
of many kinds available. Also, the protein may not like high salt ! If you
have on-column denaturation, you may never see your protein again unless you
elute with guanidine (positive side - it 'always works', negative side -
refolding is usually a nightmare, although for a hyperthermophile it's not
so bad). If in a test-tube experiment a small sample of your protein does
NOT elute off the blue with guanidine, then you probably have some sort of
real chemistry going on (it may be interesting, too).
Question: what previous steps were taken ? Did 'cooking' the sample help ?
Sometimes 'cooking' causes the thermostable protein to aggregate and to
become useless for crystallography (although often still very active !).
Does it help to use dye resins in relatively concentrated phosphate buffer?
As cheap alternatives, did you consider hydroxyapatite and / or finely tuned
Q or DEAE ?
Hope it helps. Good luck,
"D.K." <dk at no.email.thankstospam.net> wrote in message
news:9p32fb$cc2$1 at news.doit.wisc.edu...
> 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
> >E.coli. Affi-Gel Blue was found to be most suitable for chromatography
> >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
> >of using NAD+ as elutant.
> >Any idea, help, suggestion, reference (as I'm a novice in the field)
> >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.
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