Co-Immunoprecipitation (co-IP) Protocol on How to Find Protein-Protein Interactions

washing buffer
20mm Tris
0.2% Triton
or
1M Tris 1mL
20% Triton 500uL
water up to 50 ml

elution buffer
the same solution as washing buffer, but with 4% SDS (may also PI if necessary, but it may not be necessary, because the other protein has been purified out)

  1. Spin down cell debris in right ultracentrifuge at 100,000 x g for 10 minutes.
  2. If you have sufficient sample, take enough samples to be able to run a fraction of the pre-column three times in the gel (e.g.100uL)
  3. incubate the sample in sufficient quantities of beads that have been covalently conjugated to antibodies
  4. if completed binding beads / antibody is required, incubate overnight
  5. spinning down the beads for a few seconds in a centrifuge, and take some samples to run the fraction bound to the gel
  6. resuspend beads remaining in the solution and place into a rotating column (for example, from Millipore having a pore size filter from the right that prevents beads from rotating to the bottom of the tube)
  7. The supernatant spun for a few seconds and throw out the liquid that comes out the bottom of the filtrate
  8. put the column on ice
  9. add in wash buffer to the column (or, it may be necessary to wash the tube that the sample comes from the wash buffer and use it to place on the column)
  10. wash out the column for at least 5-6 times by repeating the rotating and adding new wash buffer (some people keep wash buffer to analyze whether the protein was eluted during every step of it)
  11. before adding the elution buffer, spin down and remove wash buffer
  12. If using SDS in the elution buffer, shake at room temperature (~ 250rpm) for ~ 15 minutes
  13. spun down, keeping the eluted buffer (which contains your sample), adding more elution buffer rocked another 5 minutes
  14. spun down again into the pool with the previous elution; Additional shakings may be carried out if necessary
  15. The sample run on through SDS-PAGE and perform in-gel digestion followed by mass spectrometry analysis, or Western blot to identify proteins
  16. The negative control should also be included using beads of different antibody conjugation to ensure that the co-immunoprecipitated proteins that actually interact with one another