Re: [ccp4bb]: membrane prot x-tal problem

[Edward Berry <eaberry@lbl.gov>]

***  For details on how to be removed from this list visit the  ***
***    CCP4 home page http://www.dl.ac.uk/CCP/CCP4/main.html    ***

Dear Stephen, Songlin,
This is not specific for membrane proteins, but one thing to check 
would be whether vapor diffusion is having the wrong effect. 
Try sitting drops in glass wells and seal with a well-greased 
cover slip either immediately after setting up (batch method)
or as soon as crystals start to form (interrupted vapor diffusion).

Continued vapor diffusion can dissolve the crystals if you are in the 
salting-in region of the solubility curve, or if you have a lot of 
glycerol to stabilize your protein and diffusion goes in reverse, 
diluting the well solution. If you have alcohol in the drops but
not in the reservoir, or the alcohol escapes through the tape seal,
that could also be a problem. 

If the crystals still dissolve when the composition of the mother 
liquor is constant, and no other crystal form or phase separation
droplets form to deplete the mother liquor of protein, 
I would guess the protein is being degraded.

Songlin Li wrote:
> i have recently beein trying to xtalize a 50kda membrane protein
> and have had limited success. using peg 4000, and ammonium sulfate
> i can get a few nice crystals. adding 1,6-hexanediol and some
> alcohol improves this. however, the crystals dissolve almost as
> quickly as they appear, after one or two days. i've tried detergent
> screens and additive screens but without success. does anyone know
> how i might stabilize these xtals? any advise on membrane proteins
> specifically would be a great help.
> 
> stephen kelly
> dept of microbiology
> univeristy of alabama at birmingham
> 
> ***********************************
> Songlin Li, Ph.D.
> Dept. Microbiology
> University of Alabama at Birmingham
> AL35294, USA
> Email: songlin@credit.cmc.usb.edu
> ***********************************

-- 
Edward A. Berry, MailStop 3-250
Lawrence Berkeley National Laboratory
1 Cyclotron Road, Berkeley, CA 94720
Phone: +1-510-486-4335
Fax: +1-510-486-6059
Jfax +1-530-323-9836 (you send fax, I receive email)
e-mail: EABerry@lbl.gov