Immunoprecipitation Pull-Down Assay Protocol Using Protein A or Protein G Beads

washing buffer

20mm Tris
0.2% Triton

elution buffer
made in the same buffer as washing buffer, but also containing 4% SDS (and also included a protease inhibitor if required)

1. centrifuge debris on the right ultracentrifuge at 100,000 x g for 10 minutes. to prevent any blockage during the pull-down test
2. If there are enough samples, take some samples to run a small portion of pre-column
3. incubate centrifuged supernatant with specific antibodies against the protein of interest (1 hour is generally good, overnight might be OK if no aggregation) at 4 degrees C
4. incubate for one hour or more at 4 degrees C composite samples in a sufficient amount of beads (usually Sepharose) who has covalent conjugation of Protein A or Protein G, which will bind specific antibody primer
5. spun down briefly beads in microcentrifuge, and took some samples to run the fraction bound to the gel
6. resuspend beads remaining in the solution and place into a rotating column (such as from Millipore having a pore size filter from the right that prevents beads from rotating to the bottom of the tube)
7. spin for a few seconds in the microcentrifuge and discard supernatant discharge out the bottom of the filtrate.

Immunoprecipitation Pull-Down Assay Protocol Using Protein A or Protein G Beads

8. put the column on ice
9. add wash buffer to the column
10. The column leach out at least 5-6 times more by repeating the rotating and adding new buffer wash (some people keep wash buffer to analyze whether the protein was eluted during every step of it)
11. (alternative method for this type of washing method is to add washing buffer to beads in Eppendorf tube, invert to mix, briefly and gently spin down the beads, and then remove the wash buffer.)
12. before adding elution buffer, spin down and remove wash buffer
13. If using SDS in the elution buffer, shake at room temperature (~ 250rpm) for ~ 15 minutes
14. spun down, keeping the eluted buffer (which contains your sample), adding more elution buffer rocked another 5 minutes
15. spun down again; Additional shakings may be carried out if necessary
16. analyze the various fractions obtained in the pull-down test by Western blotting (remember that one of the bands you’ll be out of the monoclonal antibody was eluted off)
17. The negative control must be included during the immunoprecipitation procedure using beads of different antibody conjugate to ensure hat the co-immunoprecipitated proteins that actually interact with one another