You can try e.g.
- micro seeding
- change MPD concentration (lower MPD especially with seeding)
- scan the pH range
- change protein concentration (lower protein conc. especially with
seeding)
- try different temperatures (4 degrees, 20 degrees, higher, if your
protein supports it)
- add another purification step
- collect data and check whether you can solve the structure with the
twinned data
- carry out restricted proteolysis or slightly modify the protein
(expression system, purification tags ...)
For your second question, you can use small volume concentrators to check
the effect of
- different salts
- different salt concentrations
- different buffers
- different pH
- additives, especially reducing agents like DTT, beta-ME
- addition of, say, 5% glycerol
I am sure there are a lot more possibilities. If you explain a little more
what you have already done, we might give even more concise answers.
Cheers, Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Fri, 21 Dec 2007, shivesh kumar wrote:
Dear all,
I have crystallized a protein in 63% of MPD at pH 3.8-4.6.The crystals are
twinned but with sharp edges.I welcome all the suggestions to get single
crystals.Also,I have needles of other mutant protein,the problem is during
concentration,it gets precipitated even in the centricon.What should I do to
avoid this precipitation to have a concentrated protein.Thanx in advance for
the suggestions.
Shivesh
