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

Biochemistry, Molecular Biology, and Cell Biology Protocols  >> Immunoprecipitation Pull-Down Assay Protocol Using Protein A or Protein G Beads

Wash buffer
20mM Tris
0.2% Triton

Elution buffer
made in the same buffer as the wash buffer, but also contains 4% SDS (and also include protease inhibitors if required)

1. centrifuge the debris in a proper ultracentrifuge at 100,000 x g for 10 min. to prevent any clogging during the pull-down assay
2. if there is enough sample, take out some sample to run a pre-column fraction
3. incubate the centrifuged supernatant with a specific antibody against the protein of interest (1 hour is generally good, overnight may be OK if there is no aggregation) at 4 degrees C
4. incubate for another hour or longer at 4 degrees C the combined sample in a sufficient quantity of beads (usually Sepharose) that have been covalently conjugated to Protein A or Protein G, which will bind the primary specific antibody
5. spin down the beads briefly in a microcentrifuge, and take out some sample to run the unbound fraction on a gel
6. resuspend the beads in the remaining solution and place into a spin column (such as from Millipore that have filters of the correct pore size that prevent the beads from being spun to the bottom of the tube)
7. spin down for a few seconds the supernatant in a microcentrifuge and dump out the liquid that comes out the bottom of the filtrate
8. place the column on ice
9. add wash buffer to the column
10. wash out the column at least 5-6 more times by repeating the spinning and adding new wash buffer (some people save the wash buffer to analyze if any protein has eluted during that step)
11. (an alternative method to this type of washing method is to add wash buffer to beads in an eppendorf tube, invert to mix, briefly and gently spin down the beads, and then remove the wash buffer.)
12. before adding the elution buffer, spin down and remove the wash buffer
13. if using SDS in the elution buffer, shake at room temperature (~250rpm) for ~15 minutes
14. spin down, keep the eluted buffer (which contains your sample), add more elution buffer to shake another 5 minutes
15. spin down again; additional shakings may be carried out if necessary
16. analyze the various fractions obtained in the pull-down assay via Western blotting (remember that one of your bands will be from the monoclonal antibody eluted off)
17. a negative control should be included during the immunoprecipitation procedure using beads conjugated to a different antibody to ensure hat the proteins co-immunoprecipitated are truly interacting with each other

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