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
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
University of
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
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
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 deterg
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
***
Ke
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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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%).
What exactly is your question--I saw tons of "crystals" of DDM and
PEGs, I think especially P400, if I recall correctly.
JPK
On Wed, Jan 11, 2012 at 7:12 AM, Patrick Loll wrote:
> Does anyone have any experience with formation of crystals of dodecyl
> maltoside in the presence of PEG?
> Pat
>
>
> ps. in passing, it seems like it would be a great idea to get together an
> excel database of all false-positive results (e.g. phosphate salt crystals)
> commonly found in the usual crystal screens. One could then search it to see
> whether one's current crystallization conditions have be vill
Jacob,
I doubt that the spherulites formed in the presence of detergents are
"primary" detergent phenomenon. However, detergent micelles and the
protein-detergent aggregates (please don't say protein-detergent
micelles) will condense (phase) out in the presence of high salt or
polymers t
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