I have always been a fan of oil, which has already been suggested. Have
you tried that?
Cross-linking has already been suggested, and these are some good protocols:
Lusty (1999) J. Appl. Crystallogr. 32, 106-112.
McWhirter, et al. (1999) PNAS USA 96, 8408-8413.
In the latter paper the crystals cracked immediately upon breaking the
seal on the cover slip (limbo trays). The cross linker was introduced
with a Hamilton syringe via a pre-cut and grease-filled hole in the
crystallization chamber. That way there were no mechanical vibrations
at all. You do NOT need to add it directly to the drop. Gluteraldehyde
has sufficient vapor pressure to permeate slowly into it. After a few
days, the crystals were incredibly robust, and gave the best
diffraction. The only problem with gluteraldehyde is if you have
primary amines in your buffer, such as tris. If that is the case, you
can usually substitute bis-tris, or just use a different kind of
crosslinker.
Another trick I like if you have a "cryo" component in the mother liquor
(protein counts) is to just let the drop dry up slowly. You can keep
"sampling" it with a small loop (removes ~1 nl) until you see it
flash-cool clear. Then, if the crystals survived, you can flash-cool
them in the dried-down mother liquor. This worked for me once with
crystals that just didn't want to transfer into anything.
-James Holton
MAD Scientist
On 10/26/2011 9:46 AM, Leonard Thomas wrote:
Hi All,
I have run into a very sensitive crystals system when it comes to cryo
protecting them. I have run through the usual suspects and trays are
going to be setup with a cryo protectant as part of crystallization
cocktail. The one problem that seems to be occurring is that the
crystals crack as soon as they are transfered out of the original
drop. I am running out of ideas and really would love some new ones.
Thanks in advance.
Len
Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251
lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571
