Re: [ccp4bb] DDM

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

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   - Protein
   - Peptide
   - Amino Acids

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

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Subject: Re: Unable to reproduce robot tray hits in hand trays
From: "Richardson, Brian C {- dot -} " bcr39 {- at -} CORNELL {- dot -} EDU
Date: 2012-03-27

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From: Jayashankar s {- dot -} jayashankar {- at -} GMAIL {- dot -} COM
Date: 2012-03-27

Subject: Re: DDM
From: Daniel Picot Daniel {- dot -} Picot {- at -} IBPC {- dot -} FR
Date: 2012-03-27

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
>

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

Previous message:
Subject: Re: Unable to reproduce robot tray hits in hand trays
From: "Richardson, Brian C {- dot -} " bcr39 {- at -} CORNELL {- dot -} EDU
Date: 2012-03-27

Next message:
Subject: Clustal omega
From: Jayashankar s {- dot -} jayashankar {- at -} GMAIL {- dot -} COM
Date: 2012-03-27