Summary: Getting better xtals

Yoram Puius puius at telico.bioc.aecom.yu.edu
Wed Aug 16 09:03:49 EST 1995


At the request of Miri Hirshberg, I'm posting a quick, low-detail summary of
the rational advice I got for getting better xtals out of stellate balls of
needles.

General categories are as follows:

1)  Protein purity

Check by SDS-PAGE
Check by IEF (isoelectric focusing)
Centrifuge protein to remove aggregates

2)  Paranoia in xtallization ;) [actually, good but uncommonly-followed advice]

Filtering xtallization solutions
Blowing dust off of cover slips (some people rationalize "The dust is good for
  nucleation!")
Be sure the precipitant is properly buffered to the pH you think it is.

3)  Change protein concentration [may nucleate too fast if too concentrated]

4)  Additives to the typical precipitants [PEG, Am2SO4],
used to slow down growth

CaCl2, MgCl2 [5-50 mM]:  Particularly good if the protein binds Ca2+ anyway,
because the cation may tighten up the structure of otherwise disordered
regions of the protein (look up the EF-hand motif, perhaps in Branden and 
Tooze.)

0.3% beta-octyl-glucoside

MPD  [try 1-10% for these]
tert-butanol
DMSO
Glycerol
Spermidine


Pardon the lame, incomplete list, with many omissions.  I'm currently trying
a number of these with little success, but hey, this is crystallization, the
totally irrational part of what we do for a living.  Many thanks to the people
who sent me all of this useful advice.


-- 
_____________________________________________________________________________
Yoram A. Puius                            Albert Einstein College of Medicine
puius at telico.bioc.aecom.yu.edu            Department of Biochemistry
M.D.-Ph.D.    Class of 1999 [?]           Bronx, NY  10461
_____________________________________________________________________________





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