Hi Joe,
It is often useful to prepare in parallel drop without protein but with
a large excess of detergent and protein buffer: a typical membrane
protein of ca 100 kDa will bind something like 100-200 molecules of DDM,
If you want to match the quantity of detergent that is bound to a 10
mg/ml protein solution, this amounts to ca 10-20 mM DDM and this does
not take into account free micelles and micelle concentration that often
occurs during ultrafiltration. In addition, even if beta-DDM crystals
could be difficult to obtain, cristalloid formation can be promoted by
lipids that copurify with protein, this can be checked by thin layer
chromatography. One also should not forget lessons from soluble protein:
spurious amount of phosphate, etc. is often easier to crystallise than
DDM or membrane protein even with crystallisation conditions supposedly
designed for membrane protein.
HTH
Daniel
Joe a écrit :
Hi all,
I am doing initial crystallization screening for a membrane protein
purified in DDM. The actual concentration of DDM in the protein
solution is >> 2 CMC after the protein concentration step (Amicon MWCO
50 kD used). I just found over 50 conditions out of 1000 give me
crystals. Having worked on soluble proteins, frankly they all look ugly
to me to different degrees. I have selected a few conditions to go
after, hoping that they will grow big enough for me to verify the
crystal identity by gels. I wonder if there is a quick way to tell if
they are detergent crystals. I know a microscope with UV capacity would
do the job, but we probably will not get one very soon. I have read
through all threads on CCP4BBS regarding detergent crystals. I have an
impression that it is actually not easy to crystallize
detergents--please correct me if I am wrong. Your thoughts and comments
are helpful.
Thank you for your attention.
--
Best regards,
Joe
