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Hi, everyone:
Thank all of your nice and quick suggestions. I would like to summarize
these oppinions as follows:
1. Transfer the Xtal to high pH as suggested by Uli, Maria, Joerg,
Armin, Kate, Rob, Lewis, Anthony and Philip. Louis suggests to increase
the precipitant concentration while increase the pH gradually.
2. Crosslinking with glutardialdehyde as suggested by Lewis, Palm and Tobias
Both Palm and Tobias recommend same the reference:
A gentle vapor-diffusion technique for cross-linking of protein crystals
for cryocrystallography
C. J. Lusty
J. Appl. Cryst., 1999, 32, 106-112
Case suggests this method as well and shares a very detail protocol as
below:
if the xtals are unstable at pH4.0 then try mild glutraldehyde
crosslinking to first stablize the xtals before transfer. It's pretty
easy to do. If you have hanging drops then transer the coverslip to a
well designed for sitting drops. Use 5 microlitres of Glut in the sitting
drop island and allow it to vapor diffuse into the drop. 0.5 to 6 hours
is sufficient. Glut relies on lysines primarily so the # of lysines and
temperature will be two major factors influencing crosslinking time.
Amines will interfere with Glut so ammonium sulfate or tris buffer would
have to be washed out and substituted before crosslinking was attempted.
3. Try other homologs as suggested by Tommi and Poul
4. Mutation as suggested by Tommi and Bernhard:
Tommi suggests to mutate the negative residues combined with pKa
calculation.
Bernhard suggest directed evolution by random mutagenesis as so on.
Thank you very much again.
Best regards
Lei
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