question: occupying protein with phospholipid?
khatipovNO at NOuchicago.edu
Wed Aug 14 12:24:25 EST 2002
You could incorporate your protein in phospholipid liposomes immediately
after purification. There are several ways to produce liposomes of various
sizes. For small liposomes just sonicate the phospholipid in the buffer,
then add protein an either briefly sonicate again or vortex. For big
liposomes, dissolve the lipid in methanol or chlorophorm, poor into glass or
plastic tube (lipid/protein ration should be determined experimentally), dry
completely so that thin film is formed on the walls of the tube, add your
protein solution and vortex ~1min on medium.
The question is would having you protein immobilized in liposomes would be
acceptable for your downstream applications.
Another possibility could be using buffers with non-polar organic solvents
(methanol, acetonitrile, ...). Again, depending on the downstream
<mmryan at med.unc.edu> wrote in message
news:20020812163328.20104.qmail at ww02.hostica.com...
> I am presently involved in purifying a protein using bacterial expression
and a His tag system. My problem occurs after I have the protein purified.
I need to occupy my protein with a phospholipid to which the protein has an
affinity for and it is at this step that my protein crashes out of solution.
My protein is generally in lysis buffer (phosphate) or lysis w/ imidazole
from the His tag elution. This protein behaves well in other buffers as
well. I can not use denaturing conditions.
> Any thoughts or comments?
> Thanks, Madge
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