Affinity purification and mass spectrometry analysis

Protocol for Co-immunoprecipitation

1. Grow 50 to 200 mL of cells in exponential phase (0.5<OD<1.0) in desired medium.

2. Harvest cells by centrifugation at 4000 rpm for 5 min at 4°C.

3. Discard medium and wash cell pellets with ice-cold 1X PBS. Centrifuge at 4000 rpm for 5 min at 4°C.

4. Discard supernatant and resuspend cell pellets in 2 mL ice-cold lysis buffer (6 mM Na₂HPO₄, 4 mM NaH₂PO₄, 150 mM NaC₂H₃O₂, 5 mM MgC₂H₃O₂, 0.25% NP-40, 2 mM EDTA, 1 mM EGTA, 5% glycerol, 2 mM benzamidine and complete EDTA-free protease inhibitor cocktail). Slowly drop suspension into liquid nitrogen to form “pop-corn”. Samples can be stored at -80°C at this stage.

5. Perform cell lysis using Ball Mill apparatus (Retsch, MM400): place “pop-corn” in appropriate cylinders and perform 5 cycles of 3 min at a 10 Hz frequency, cooling cylinders in liquid nitrogen after each cycle. Then, recover frozen powder in 15 mL Falcon tubes. Samples can be stored at -80°C at this stage.

6. Thaw frozen powder in ice and transfer to 2 mL eppi. tubes.

7. Clear extracts by centrifugation at 10000 g for 10 min at 4°C. Transfer supernatants to new 1.5 mL eppi tubes.

8. Save a 40 µL aliquot for Whole Cell Extract (WCE) analysis. 

9. Add 1 mg of pre-washed antibody-conjugated M-270 Epoxy Dynabeads (Invitrogen, #14311D) for immunoprecipitation and incubate on a wheel (20 pm) for 1 hour at 4°C.

10. Perform 2 washes with 1 mL ice-cold IPP150 (10 mM Tris pH8, 150 mM NaCl, 0.1% NP-40).

11. Boil immunoprecipitated and WCE fractions at 95°C for 10 min in the presence of sample loading buffer and analyze samples by SDS-PAGE Western blotting using appropriate antibodies.