William Klimke wrote:
>> I am planning on using immunoprecipitation to determine protein-protein
> interactions of an outer membrane protein in Escherichia coli. I know that
> they have used IPP to pull down proteins in mitochondrial membranes. Does
> anybody have, or know of, any useful protocols that I might be able to use.
I like to use dead Staph. aureus cells (Pansorbin, Calbiochem) for IPs.
You add a small amount of the suspension to your sample, incubate on ice
and spin. The pellet can then be resuspended directly in SDS sample
buffer. Calbiochem produces a handy (and free) booklet on the use of
Pansorbin.
Before you can precipitate, however, you'l have to isolate and
solubilize the membranes from your E. coli cells, without breaking
protein-protein interactions. There is no way to predict which
conditions will be optimal for your particular proteins. Mild detergents
like dodecylmaltoside, octylglucoside, C12E8, hecameg or similar may
work. Sometimes you can also get away with Triton-X100, which is
cheaper. Increase the detergent concentration, starting from the cmc, at
a const [protein] of 1-2 mg/ml and see at which concentration your
protein becomes solubilized, without breaking protein-protein
interactions. The whole thing unfortunately is black art, not science.
Good luck!
