Re: [ccp4bb] OT: Crystallisation compatible detergents

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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From: Engin Ozkan eozkan {- at -} STANFORD {- dot -} EDU
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Subject: Re: OT: Crystallisation compatible detergents
From: Pascal Egea pascal {- at -} MSG {- dot -} UCSF {- dot -} EDU
Date: 2009-03-24

Hi Darren
I believe that the most frequently used detergent for protein
crystallization (not including membrane proteins) is octyl-glucoside.
The most important parameter is the CMC of the detergent and the size of the
micelles of free detergent if you have micelles around. These considerations
do not apply if you are well under the CMC (only free molecules of detergent
are present in solution). I believe tween-20 CMC is roughly 0.006% so you
are at the border.
The problem is you have micelles of detergent is that depending on their
size and the MW cutoff of the device you use for protein concentration you
may end up concentrating both components protein and detergent and then this
is when trouble starts. You may have an unknown amount of concentrated
detergent above the CMC, this can be very problematic for crystallization
because you may have a lot of phase separation in your drops; phase
separation are usually (but not always) not desirable.

For membrane protein crystallization we are always facing this problem
because we usually work in this regime of critical detergent concentrations.
However for soluble proteins that need a little bit of detergent to remain
stable this is not as usual.

OG (octyl glucoside) is a rather mild detergent with a CMC of about 20 mM
(check ANATRACE catalog).
To simplify, maybe a little bit too much, the most important is at first to
find the minimum amount of detergent you need to keep you sample stable and
possibly stay as low under the CMC. And then may be go up in detergent
concentration if you don't get the results expected. You can check that your
complex stays functional using Biacore (in your specific case)
Other detergents very popular for non membrane proteins are DDM (dodecyl
maltoside) or CHAPS (zwitterionic cholesterol derivative).
So in order of decreasing preference I would suggest OG, CHAPS (which is a
totally different type of detergent) and DDM (this one has a very low CMC
~0.15mM I believe, so it is difficult to get rid of it, but it is fairly
genlte).
An alternative to detergent are non-detergent sulfo-betaines they can
sometime have the same protective effect without the trouble of detergents.

I hope this helps,
Cheers

Pascal F. Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics

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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: two identical proteins in one asymmetric unit
From: Engin Ozkan eozkan {- at -} STANFORD {- dot -} EDU
Date: 2009-03-24

Next message:
Subject: Re: pins - was Re: images
From: James Holton JMHolton {- at -} LBL {- dot -} GOV
Date: 2009-03-24