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



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Dear Stephen,

there are many things to fiddle with and it can become quite desperate. 
Some (maybe trivial) points to consider are:
*	if you use sitting or hanging drop not only protein and precipitant
concentration rises but also that of the detergent => could this dissolved
your protein Xtals ?  you might want to start with as few detergent as
possible
*	are they protein or salt or detergent xtals ? You can in priciple
also obtain some quasi crystalline detregent phase (those "crystals" often
look very roundish and colourful)
*	can you freeze them at least for characterization (see the
oil-freezing discussion on the BB if you have no cryoprotectant) . there´s
microfocusbeamtime out there if they are too small for the home source
*	how did you do the detergent screens? remind you that its often
difficult to do a complete detergent exchange via dialysis. ion exchange
chromatography is more reliable.
*	how about other techniques as lipidic cubic phases (used for
bacteriorhodopsin, Landau et al. in PNAS)
*	if you get really desperate and have some idea where your protein
sticks out of the membrane, you can mutate potential surface residues
(worked for some of us but is rather tedious, Pautsch et al in Proteins, Vol
34).

good luck!


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Dr. Alex Pautsch
 Protein Crystallography /Structural Research 
 Boehringer Ingelheim Pharma Deutschland 
 Birkendorferstrasse 
 88400 BIBERACH, Germany 
 tel. +49 - (0)7351 - 54 4683 
 fax. +49 - (0)7351 - 54 98390 
 email alexander.pautsch@bc.boehringer-ingelheim.com
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> -----Ursprüngliche Nachricht-----
> Von:	songlin@credit.cmc.uab.edu [SMTP:songlin@credit.cmc.uab.edu]
> Gesendet am:	Mittwoch, 14. Juni 2000 18:34
> An:	ccp4bb@dl.ac.uk
> Betreff:	[ccp4bb]: membrane prot x-tal problem
> 
> ***  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 users,
> 
> 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
> ***********************************