DDM

From: Katarzyna Rudzka
Date: 26 March 2012 18:17

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

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From: yybbll

Hi,

I used 0.017% or 0.012%. My protein is very stable at this concentration.

Good luck.

Lin

2012-03-26

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yybbll

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From: Kelly Daughtry
I generally use 1 - 2% DDM for extraction only, but lower the concentration to 0.01% for following steps (i.e. NiNTA and gel filtration). The excess DDM is washed away by using a lower concentration in your wash and elution buffers.

Kelly

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From: Das, Debanu
Hi Katarzyna,

Yes, membrane proteins can be purified in DDM at ~1-2xCMC. Since its CMC value is very low, at the extraction step you need to use a much higher concentration up to ~100x CMC. During different rounds of purification, you can bring the CMC level down to 1-2x CMC and even try detergent exchange in the last step, although it can be difficult to totally exchange out the DDM due to its low CMC. Also use a 100 kDa MWCO concentrator since the DDM micelle size is large.

In general, DDM is a reasonable choice for extraction, purification and crystallization setups, but it will be worthwhile to screen a panel of different detergents for extraction and purification for your particular target.
In 2005, we set up a fast protocol for screening a panel of 18 detergents in 48-72 hours, http://smb.slac.stanford.edu/~debanu/posters/012605_PPCW2005_DDAS.pdf, which includes suggestions for starting concentrations for extraction.

There are also some papers on detergent screening, for example:
http://www.ncbi.nlm.nih.gov/pubmed/18988031
http://www.nature.com/nprot/journal/v4/n5/full/nprot.2009.27.html (Stroud lab)

Thanks,
Debanu.

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From: Joao Dias

Kasia,

A lot of people uses DDM to purify membrane proteins, not a lot of people crystallises them.

If you want to crystallise a protein purified in DDM, then you should use LCP.

If you go for vapour diffusion, you should exchange the DDM for a detergent with a smaller micelle size otherwise you might get crystals but it is difficult to get good diffraction. Try mixed micelles for example.

Typically use 0.05% DDM during purification and use 100kDa cut-off membranes in order to prevent detergent concentration.

For extraction it depends on your protein and expression system but you can see in the literature values between 0.5-2% being used successfully.

Good luck.

Cheers,

Joao

Joao Dias, Ph.D.

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From: Edward A. Berry
If I recall correctly cytochrome oxidase, which I believe was
the first protein purified with DDM, requires about 10x cmc
in column buffers to keep it soluble. Check for papers from 1970's or 80's
by S. Ferguson-Miller.

Cytochrme bc1 complex, on the other hand, is perfectly clear in 1 cmc
and can be diluted from that into detergent-free buffer for
spectroscopywithout becoming turbid, at least within an huor.

My rule of thumb for solubilizing with DDM is 1 g/g protein (+ 1 cmc).
If protein is say 10-20 g./l, the (+ 1 cmc) becomes completely insignificant.
(Total protein, not the protein of interest. Probably depends on the
amount of lipid too, but this is just a rule of thumb to start with.)

eab

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From: Ho Leung Ng
Actually, DDM is the most successfully used detergent for membrane
protein crystallization. See Newstead et al, Protein Sci. 17:466. But
yes, the rule of thumb is that detergents that form smaller micelles
give better diffracting crystals, but are more destabilizing.


Ho

Ho Leung Ng

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From: Daniel Picot
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