Co-Immunoprecipitation

[aftabac]

Hi
there are several ways to find out protien protein interaction and for this the gold standard procedure is Yeast two hybrid assay, and now many other methods are also investigated to find out protein protein interaction. one of the modern used technique is Co-immunoprecipitation whcih can be defined as

"A purification procedure to determine if two different molecules (usually proteins) interact. An antibody specific to the protein of interest is added to a cell lysis. Then the antibody-protein complex is pelleted usually using protein-G sepharose which binds most antibodies. If there are any protein/molecules that bind to the first protein, they will also be pelleted. Identification of proteins in the pellet can be determined by western blot (if an antibody exist) or by sequencing a purified protein band."

more comments..........

regards
aftab

[oBWhat]

Hey Aftab,

yeah I think this is a great topic to cover. More on protein-protein interactions, I guess that more modern techniques would also include FRET and luciferase monitoring on protein-proteni interactions.

Both of these can be automated for high-throughput analysis of protein-protein interactions. Also dont forget proteomics for the analysis of protein-protein interactions by using the presence or absence of cross-linking, I think that is the FUTURE of protein-protein studies!

[GRRLyeast]

this method is not working for me i dont get any results. And the protein i am using as bait is a membrane protein and i cant seem to get the results. Any suggestions?

[kevin_a]

Working with membrane proteins in general can be tricky.
Once you take a membrane protein out of the membrane and expose all it's hydrophobic bits to buffer, things may not work correctly.

Can you describe your CoIP protocol a little more? Lysis buffers and conditions. Any chance the capture antibody binds to the site of protein interaction or just generally inhibitory. Is the pull-down of the membrane protein working (does it show up on a Western).

If you are a least getting the membrane protein to pull-down and show up on the Western, you can try cross-linking the prey protein(s) to the bait prior to the lysis allowing you some more options in lysis buffer conditions. Cross-linker of choice will depend on if your interaction is taking place inside or outside of the cell.

K

[keithbrent2000]

Somtimes it can depend on the detergent that you use. Some detergents may disrupt your protein complex more than others. You can try to vary your detergents and also the concentration of the detergent. Some common ones to use include Triton X-100, octylglucoside, and CHAPS.