Hello Balaji,
You may try the following:
1) Try different detergents
2) Use a 100 kDa concentrator after protein purification if possible, to
eliminate as much of empty detergent micelles as possible. You probably
have a lot of these since the CMC value of LDAO is ~0.023% and mol. wt.
of LDAO micelles should be ~17 kDa.
3) LDAO conc. down to ~1-2x CMC
4) Additive screens
5) Purification by SEC before crystallization
Thanks,
Debanu.
Bhyravbhatla, Balaji wrote:
Hello All,
We are trying to crystallize a membrane protein but cannot get the
xtals to grow bigger. Presently we have only thin needles (diffracting
to about 8A). Thus far we have tried to change protein concentration,
LDAO concentration, PEG screen as well as temperature. Have tried
macro and micro seeding as well.
A typical setup looks like: 10% PEG 6000, 0.1M Citrate 5.5, 0.1M
Li2SO4 and 5% MPD with about 12mgs/ml of protein which has about 0.2% LDAO
Any suggestions or improvements that we can try would be appreciated.
This construct of the protein is not in the membrane but membrane
associated.
Thanks
Balaji
