Markus Zettl wrote:
> > I solubilized membrane proteins by Triton X-100 and for further
> > aplications I want to get rid of this detergent. Could you tell me a way
> > to remove Triton X-100 from my probes leaving membrane proteins in
> > native conformation.
Simply removing the detergent would lead to protein precipitation in
most cases. There are two ways forward: Exchange against a different
detergent which does not interfere with further processing or
reconstitution into proteoliposomes.
Detergent exchange is usually accomplished by binding the protein to a
chromatographic column (hydroxyapatite, affinity...) and then washing
extensively with a solution of the new detergent. Then the protein is
eluted, the elution buffer of cours must also contain the new detergent.
Reconstitution into proteoliposomes is achieved by adding lipid (soy
bean asolectin is cheapest, but does not suffice in every case),
dissolved in detergent solution. A starting point for optimisation would
be something like 1 mg/ml protein, 5-10 mg/ml lipid. Then the detergent
is slowly removed, so that the lipid formes liposomes (vesicles enclosed
by a double membrane), into which the protein is integrated, hopefully
as it would be in a natural membrane.
Detergent removal is usually obtained by one of three different methods:
Dialysis, gel filtration and binding to hydrophobic gels. Because Triton
X100 has a very low cmc, you will not be able to use the first two
methods, which leaves you with the third. Biobeads 5M2 are the most
frequently used (BioRad). They need to be washed in water, 1 M acetic
acid, 50% methanol, 100 % methanol, methanol/aceton, 100% aceton and
then the whole series backward before use. Add beads to your sample, mix
for 30 min, spin and repeat until all detergent is removed. For
occasional experiments you can also use chromatographic columns
prepacked with such gels (for example Pierce), but they are too
expensive for routine use.
You may wish to use detergents with high cmc like Octylglucoside, in
which case all you need to do is to dialyse your sample against
detergent free buffer (8h and 16 h against 1000 times the volume of your
sample) for reconstitution.
Good luck (you'll need it <G>)!
