It is important to distinguish between the solubilisation and the
purification steps:
1) During the solubisation step you need to care about the
lipid/detergent ratio. The amount (not the concentration) of detergent
(i.e. in non monomeric form, the detergent above the cmc) is important.
You may need a high amount of detergent.
2) During the purification step, you need to keep your protein soluble.
Here, the concentration is important and you may keep the concentration
of detergent around the cmc. But you have to be aware that in the
initial (and also the not so initial ones!) steps you may have a lot of
lipids, you need then keep the concentration of detergent fairly high in
order to keep everything soluble. I like to decrease the detergent
concentration at each purification steps in order to avoid protein
denaturation. The protein may sustain a fairly high detergent
concentration of detergent during the early steps since the
lipid/detergent mixed micelles will be less denaturing than the pure
detergent micelles in the later steps. Thin layer chromatography is a
quick and easy method to check if you have a large amount of lipids in
your preparation.
HTH
Daniel
Le 26/03/2012 19:17, Katarzyna Rudzka a écrit :
Hi All,
Has anyone had any luck purifying membrane proteins with DDM
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ?
(Its CMC is very low: 0.009%). I would like to keep it as low as
possible, so I don't have too much DDM around when I get to the
crystallization step. I wonder If the amount of detergent sufficient for
the protein extraction has to be determined experimentally for each
protein or maybe there are some good rules of thumb. I appreciate your
help. Thanks.
Kasia
Katarzyna Rudzka, Postdoctoral Fellow
Department of Biophysics and Biophysical Chemistry
Johns Hopkins University, School of Medicine
Baltimore, Maryland 21205 USA
