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This protocol uses total cell extracts to analyze putative protein-protein interactions in eukaryotic cells. One can also use nuclear extracts as a source for this protocol.

  1. Transfect 2-T150s of HEK293 cells with the appropriate expression vectors (protein A and protein B) as per standard protocol. Alternatively, you can use an endogenous cell line that expressed both proteins if you have specific antibodies for each protein.
  2. After 48 hours, harvest cells and wash with PBS -/-
  3. Transfer cells into 2 microfuge tubes and lyse each tube of cells with 1ml of:
  4. 20 mM Tris pH 7.5

    100 mM NaCl

    0.5% NP-40

    0.5 mM EDTA

    0.5 mM PMSF

    0.5% protease inhibitor cocktail (Sigma)

    pipet cells up and down extensively and then sit on ice for 10 minutes.

  5. Spin down cell extracts at max speed for 10 minutes at 40C.
  6. Pool extracts from each transfection into a 15 ml tube. For immunoprecipitation, add 10 ml (polyclonal serum) or 10 mgs (monoclonal antibody) to 1 ml of extract. Incubate with end-over-end mixing for 2 hours at 40C. Save 50 ml of each cell extract and add 50 ml of 2X SDS-PAGE sample buffer and boil for 5 min.
  7. Wash 50 ml of Protein A/G Sepharose (Santa Cruz Biotech) with 1 ml of lysis buffer---3 times and then remove last of excess buffer carefully.
  8. After the 2 hour incubation (Step 5), add the washed Protein A/G Sepharose to the immunoprecipiates and mix end-over-end for 1 hour at 40C.
  9. Wash immunoprecipitates 3X with 1 ml of lysis buffer and remove last of buffer carefully. Add 50 ml of 2X SDS-PAGE sample buffer and boil 5 min. Load on SDS-PAGE gel as follows:
  10. Lane 1 PAGE standards

    Lane 2 protein A transfected cells

    Lane 3 protein B transfected cells

    Lane 4 protein A/B transfected cells

    Also run a gel with just the extract (Step 5) before immunoprecipitation to show that each cell line expresses the right protein.



  11. Western blot get to Immobilon membranes (Millipore) and block in the following buffer overnight:
  12. Blotto

    50 mM Tris pH 7.5

    100 mM NaCl

    0.1% Tween 20

    5% dry milk

  13. Probe the next day with the appropriate antibody combination and develop using the ECL kit (see Western blot protocol)