Dear Bei, You question is not off topic, no apology needed.
Have you considered other possibilities than using detergents? A little bit of glycerol might help, or e.g. a different salt, pH, buffer, some additive, or simply salt concentration. I once met a protein which was not soluble much beyond 1-2 mg/ml below 300mM NaCl and with 500mM NaCl could be concentrated to more than 100mg/ml. With the little amounts you have you could also try restricted proteolysis in order to determine stable subdomains. If you want to crystallise the protein, a detergent is not really favourable and it is worth playing with the above conditions before trying detergents. Tim On Tue, Sep 07, 2010 at 03:30:04PM -0400, joybeiyang wrote: > Hi everyone, sorry for this none CCP4 question, but I am working on some > tough proteins which easily got aggregated after purification, I am thinking > of using some detergents rightnow, however, I am a newbie in this field, > could you please give me some advice on the usage of detergents? > > here is some basic information about the protein: > 1. This protein is as big as 90KD. > 2. The productivity of the protein is very low, and the homogeneity of the > protein is not good. Most of the proteins come out of the column at void > volumn, and the rest of the proteins forms 3-4 peaks, and each of them > features low peak height. 3. I have already tried different truncates of the > protein and homologous proteins from other species, up to now the protein on > my hand is the best of them. 4. I have stocks of the following > detergents:n-Octyl-b-D, n-Decyl-b-D, FOS-Choline-12, n-Dodecyl-b-D, Cymal-5, > CHAPS, Tween20, Triton-X100. > > my question is: 1. would you please recommend some other detergents to try? > 2. should I try different concentration of the detergent and how? right now I > just tried the concentration equals the CMC, some of the detergents do improve > the homogeneity of the protein, however, once I concentrate the protein for > crystallization, the detergent get concentrated too, and as you know that is > very bad for crystallization. 3. would you please recommend me some > literature to resort to (about the same situation as mine or about the use of > detergents)? > 4. any other suggestions or comments about how to improve the quality of the > protein. > > Your suggestions and comments will help me a lot and will be highly > appreciated. > > Many thanks to all of you! > > Bei > > 2010-09-07 > > > > joybeiyang -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
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