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
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Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
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