Protocol for In Vitro Double Immunoprecipitation

  1. Assemble TNT (Promega cat. #L4610) reactions:
    2. Master mix (per sample):
    6.25 µl retic. lysate
    0.50 µl buffer
    0.25 µl T7 polymerase
    0.25 µl met-aa mix
    1.00 µl [35S]-methionine (Amersham cat. #SJ1015)
    0.25 µl RNasin (Promega cat. #M2511)
    1.00 µl 12.5 mM ZnCl2
  2. Assemble DNA:
    3. For interactions between 2 proteins, use 1.5 µl of each DNA at 0.125 µg/µl (0.375 mg total).
    For interactions between 3 proteins, use 1.0 µl of each DNA at 0.125 µg/µl.
    It is recommended to use constructs from a plasmid with an optimized translation intitiation site like the pcDNA3 series (e.g. HA-tagged, FLAG-tagged, and untagged).
    Add 9.5 µl master mix to 3.0 µl DNA. Mix and incubate at 30°C for 2 h. The reaction size can be doubled if signals are hard to detect, especially for interactions involving 3 proteins.
  3. Keep buffers and samples on ice or at 4°C from now on. Add 112.5 µl FLAG lysis buffer. Remove 2.5 µl as 2 % total:
    4. FLAG Lysis Buffer: 25 mM Tris pH 7.5, 300 mM NaCl, 1% Triton X-100, 200 µg/ml ethidium bromide and freshly added protease inhibitors, usually 10 µg/ml each aprotinin and leupeptin and 1 mM PMSF.
  4. Preclear remainder with 20 µl protein A sepharose for 1 h on rotating wheel, then centrifuge 5’ max speed at 4°C:
    5. Sigma cat. #P3391- swell beads in PBS, then block at least 1 h at RT in PBS + 1 mg/ml BSA, wash several times with PBS, and store in PBS/0.02% azide at ~50% suspension. Cut pipette tips with clean razor blade for accurate pipetting.
  5. Add 1 µl αFLAG to the supernatant. Incubate 1 h on rotating wheel:
    6. Eastman Kodak M2 monoclonal antibody cat. #IB 13025.
  6. Add 20 ml protein A sepharose for 1 h.
  7. Pellet beads 1’ at 3000 rpm. Remove supernatant with vacuum trap setup.
  8. Wash twice with 1ml FLAG lysis buffer, pelleting each time 1’ at 3000 rpm, then once or twice with 1 ml FLAG final buffer (25 mM Tris pH 7.5, 140 mM NaCl). Be sure to remove all buffer on this last wash.
  9. Add 25 ml SDS lysis buffer (20 mM Tris pH 7.5, 50 mM NaCl, 1% SDS, and freshly added 1 mM DTT). Boil 3’. Quick spin to pellet beads.
  10. dd supernatant to cold 225 ml RIPA (10 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Add 2.5 ml αHA antibody and incubate 1 h on rotater again. (Mouse monoclonal HA.11- BAbCo cat. #MMS-101R-1000).
  11. Add 20 ml protein A sepharose 1 h.
  12. Pellet beads and remove supernatant as above. Wash 3-4 times with 1 ml RIPA. Suspend beads in 2X Laemmli sample buffer, boil, and run on SDS-PAGE. Soak gel at least 1 h at RT in 1 M sodium salicylate and dry gel on gel dryer before autoradiography at -70°C. 8 h to overnight is usually fine for interactions involving two proteins, several days might be required for interactions with three proteins.

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