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
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> cel: 773.608.9185
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