The carry-over would be DDM, and my guess would be DDM crystals formed
upon dehydration. The DDM concentration will be huge after repeated
dilution/concentration cycles. DDM micelles will hardly pass the 50
kDa cut-off filter so imagine how much you have added!
Poul
On 31/10/2008, at 02.21, 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]
*******************************************
