pjura at proaxis.com
Thu Nov 19 17:59:32 EST 1998
Stefan Backstrom wrote:
> Hello there.
> I have some really fragile crystals.
> How can I improve stability ??
> I know that some people have tryed glyceraldehyde, does anyone have any
> references ??
> Other tricks ??
> I have peg 3350 and glycerol in the drop.
> Stefan Backstrom
> Umea University
> s-901 87 Umea
> email: stefan.backstrom at ucmp.umu.se
> tel: +46 907856794
> fax: +46 90778007
These references for crystal crosslinking are from Blundell and
Quiocho, R. A. and Richards, F. M. (1964). Proc. Natl. Acad. Sci.
U.S.A. 52, 833 -- crosslinking carboxypeptidase crystals with
glutaraldehyde resulted in crystals having greater mechanical strength
and total insolubility in deionized water.
Lipscomb, W. N., Coppola, J. C., Hartsuck, J. A., Ludwig, M. L.,
Muirhead, H., Searl, J. and Steitz, T. A. (1966) J. Molec. Biol. 19, 423
-- crosslinking studies of carboxypeptidase A (these might be
biochemical studies of crosslinking of the free protein in solution to
determine subunit-subunit interactions, rather than crystal crosslinking
studies, but they might give you some useful information on other
crosslinking agents to try).
Bunn, C. W., Moews, P. C. and Baumer, M. E. (1971) Proc. Roy. Soc.
London B178, 245 -- crosslinking calf rennin crystals to stabilize them
for transfer to low-salt conditions for derivitization.
Richards, F. M. and Knowles, J. R. (1968) J. Molec. Biol. 37, 231
Korn, A. H., Feairheller, S. H. and Filachione, E. M. (1972) J. Molec.
Biol. 65, 525 -- these two discuss the chemistry of the reaction with
In addition to these references, you might look into the biochemical
literature on the determination of subunit-subunit interactions in
multimeric proteins, which relies on the use of crosslinking agents and
gel electrophoresis, and the literature on tissue sample preparation for
electron microscopy, where extensive use is made of these reagents to
fix samples for embedding and thin sectioning.
Two points about this approach. First, it will probably take some
trial-and-error work to find the right crosslinking reagent and the
right conditions to make this work. Second, crosslinking crystals
usually decreases their resolution somewhat.
If you can use a more traditional approach to stabilizing your
crystals, such as increasing the concentration of your precipitant after
the crystals are fully grown, transferring the crystals into some other
solution in which they are more stable, cooling or freezing them during
data collection, or, best of all, finding a more stable crystal form,
this would make your life much easier in the long run than having to
crosslink your crystals. But if this is the only way that works for
you, then go for it. Either way, good luck.
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