20 mM Tris, pH 8.0 containing 0.2% Trition X100
Wash buffer containing 4% SDS (and also include protease inhibitors if required)
NOTE: The choice of wash and elution buffers should be empirically determined. The buffers listed here are for general use and will not be ideal for all proteins/systems. If phospho proteins are being pulled down the addition of a phosphatase inhibitor may be required to prevent degradation.
- Centrifuge the cellular debris in an ultracentrifuge at 100,000 x g for 10 min to remove any non- soluble material that will interfere with the later assay.
- If sufficient sample is available it is prudent to clear by means of preliminary pass or mock pull down using a pre-column or gel.
- Incubate the cleared supernatant with specific antibody against the protein of interest (1 hour is generally sufficient but an overnight incubation may be required for some Ab-Ag interactions). Incubate at 4 degrees C for O.N. or 37 C for shorter periods.
- Incubate Sample/Ab mixture with an adequate amount of protein A (or G) speharose) 1h at 4 degrees C.
- Spin down the beads briefly in a microcentrifuge, 3-5 minutes 1000g, and remove and save supernatant to assay for unbound (depleted) fraction.
Wash beads by adding wash buffer vortexing and spinning down as before. At least three cycles (preferably more) of wash are required to remove any unbound material from gel pellet.
NOTE: it may be advisable to ad 0.5 M NaCl to the wash buffer to aid in removal of any non-specific binding to the gel.
- Spin one final time – remove supernatant and retain pellet.
- Add elution buffer to the tube and vortex. Let sit on ice with periodic vortexing for 15 minutes.
- Spin down, retain the eluted material /buffer (which will contains you’re your eluted protein of interest/sample. It may be required to repeat the elution step and poll or assay elutions separately as required – depending on the avidity of the antibody for ligand and protein A/G for your Ab.
- Analyze the various fractions obtained in the pull-down assay via Total protein staining or via Western blotting (remember that your sample will contain the monoclonal/polyclonal antibody eluted off the gel and perhaps protein A and G as well – if from a supplier other than Exalpha). If western blotting is performed, it is generally accepted that a separate Ab (not the one used to pull down) should be employed).
- A negative control should be included during the immunoprecipitation procedure using beads conjugated to a different antibody to ensure that the proteins co-immunoprecipitated are true positives.