Jacob,
DDM should never crystallize under any aqueous conditions seen for
protein crystallization. We tend to store 20% stock solutions (~0.4
M) of DDM in buffer at 4C. DDM tends to phase out long before it
would crystallize (~ 0.8 mol percent).
With this said, how have you set up the storage system? How leaky are
the containers? And where did you get the DDM? While most sources
of DDM are great (e.g., Anatrace), some have poorer QC. Our commonly
used alkyl-beta-D-glycosides should not crystallize under any aqueous
conditions seen for protein crystallization, BUT alkyl-alpha-D-
glycosides will easily crystallize under most aqueous conditions (I
have worked in a 37C room trying to keep alpha-octyl glucoside from
crystallizing before my porin samples). Occasionally, some batches
of alkyl-beta-D-glycosides will be contaminated with the alpha
anomer, which could crystallize out at low temp. I have seen one
bottle of "beta"-octyl glucoside that would even dissolve at room
temp. A quick TLC of your detergent stock should show contamination
or not.
Another possibility is that the crystals are protein. With OmpF
porin in beta-octyl glucoside, crystals were obtained via
microdialysis by changing the PEG4000 from 10% to ~14%. One time, I
didn't finish setting up the microdialysis cells one evening, and I
put the remaining protein stock (with 10% PEG4000) in the fridge.
The next morning, I discovered nice porin crystals in the tube.
Alkyl-beta-D-glycoside detergent micelles tend to aggregate/cluster
at lower temperature (i.e., they have an upper consolute boundary),
which can nucleate protein crystals. Adding compounds like PEG
raises that boundary to and above room temperature, causing phase
separation and, in the case of porins, crystal formation.
Hope this provides some ideas,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [EMAIL PROTECTED]
****************************************************************
On Oct 30, 2008, at 9:21 PM, Jacob Keller wrote:
Dear Crystallographers,
has anyone heard of or seen crystals form in their membrane protein
stocks in the presence of DDM, such as from a breakdown product, or
otherwise? My protein stock, after having been stored at ~2mg/mL
(around solubility limit) at 4degC in (theoretically) nothing but
~5mM DDM and 10mM NaHEPES 7.0, formed a significant, visible number
of spontaneous crystals in the bottom of the tube (two separate,
identical aliquots) after ~5mos. I have heard of spontaneous
crystals for soluble proteins, but never for membrane proteins.
The protein was buffer-exchanged into (10mM HEPES 7.0 and 1mM DDM)
by 2 x 30-fold then 1 x 15-fold concentration-dilution in 50kD MWCO
concentrator. Seems pretty thorough to me, but perhaps there was
some kind of carry-over?
Substances encountered in the course of the prep:
HEPES
NaCl
protease inhibitor cocktail
EGTA
DNAse
TCEP
PMSF
LysoPhosphosphotidylCholine 16
DDM
imidazole
glycerol
Thanks,
Jacob Keller
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
*******************************************
