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Dear Cécile, Could you give more details about your strategy of exchanging detergent? I used to try exchanging detergent during wash step of gravity purification (so I assumed the idea is similar to yours), but most of protein aborted binding. Without exchanging detergent, the protein bound to its ligand pretty well. This phenomenon was also consistent with another experiment, in which we saw detergents had an affect on binding affinity between protein and ligand. Dear Vasan, This paper may give you useful information regarding to accumulation of detergents during concentration using concentrators with different MWCO. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2279333/ Best regards, Sally. 2010/10/7 Cécile Breyton <[log in to unmask]>: > Dear Vasan, > > 3CMC seems ok when you work with DDM, or even LDAO, but it becomes way too > much when you work with OG. > As a general rule, it is not a good idea to think in "times cmc" because the > cmc can be very different from one detergent to the next. > In this ex, DDMcmc=0,17 mM, 3cmc=0,51 mM which is reasonable. > OGcmc=20-25 mM, 3cmc=60-75mM which is far too much. > It is much more reasonable to think in terms of "concentration of detergent > above the cmc", which is what the protein "sees". > In this ex., if 3DDMcmc is ok ([DDM above cmc]=0,34mM), a [OG]= 25-26 mM > would be OK ! > It indeed is enough. You must ruin the lab doing gel filtration at 3OGcmc! > LDAO is an intermediate case (LDAOcmc=1mM in H20 but drops when salts are > added). > > Another point is that gel filtration might not be the best technique to > exchange detergents, especially DDM, which has a low cmc... You mention your > protein has a tag, the best way is to immobilise the protein, wash > thoroughly (20CV) with buffer in the new detergent, and elute with the new > detergent. Works well also with ion exchange columns. > > There was indeed a thread not long ago about the importance of the WM > cuttoff of membranes for protein concentration. The lower it is, the better > you concentrate the micelles, especially if you already have tons of them. > Remember that your protein is larger than just the protein mass: it also has > all the detergent bound to it that makes it bigger. > > HTH, > > Cécile > > > Le 06/10/10 21:57, R.Srinivasan a écrit : > > Dear all, > > We work with lipid droplet binding proteins in our group. These > proteins behave more like membrane associated proteins, have a very > hydrophobic surface and require detergents right from solubilization to > crytallization. > > We use 1% DDM for solubilization, 0.03% DDM throughout affinity > chromatography and exchange 3X CMC of OG and LDAO in gel-filtration column > for crystallization. Previously, we used to use concentrators with 30kda cut > off but recently got suggestions to use higher cut off concentrators. Our > proteins arent generally big and are less than 100kda. > > Now my question is that, we havent really got any crystals over a > one year period and is it that the 3X CMC of detergents is too much for > these globular proteins in the sense it is engulfing the protein on a whole > and blocking crystal contacts? Is there a way i could play around with > detergents getting closer towards crystals? > > Looking forward to valuable suggestions and thanks very much in > anticipation. > > Sincerely, > Vasan > > > > > >