Immunoprecipitation of ³⁵S Labeled Cells

1. Cell Labeling
   1. Start with 1-5 x 10⁶ cells that are in log phase growth
      1. For suspension cells, passage cells several hours prior to harvest for metabolic labeling. Cells that have recently been split may be in stationary phase and may not incorporate the radiolabel as efficiently as proliferating, log-phase cultures.
      2. For adherent cells, plate cells 1 day prior to metabolic labeling. Cells that are approximately 70-80% confluent are optimal, however this may vary according to cell type.
   2. Wash the cells in warm (37°C), serum-free medium (e.g., DMEM) which does NOT contain cysteine or methionine.
   3. Resuspend the cells in cysteine/methionine-free medium, containing 2% dialyzed fetal calf serum (FCS) and incubate at 37°C for 30 min (in the presence of CO₂, if normally required).

      NOTE: Follow the radioisotope safety guidelines for all subsequent handling.

   4. Add 100 mCi/ml each of L-[³⁵S]Cys and L-[³⁵S]Met to the cells and mix well. Incubate the cells for 1-4 hr at 37°C, with occasional mixing to resuspend the cell pellet.
2. Cell Lysis
   1. Wash the cells 3 times in ice cold PBS.
   2. Harvest the cells, gently scraping the plate if adherent cells are used. Centrifuge to pellet cells.
   3. Lyse cells by resuspending the cell pellet in 10 volumes of pre-chilled lysis buffer* or pre-chilled RIPA buffer* containing protease inhibitors*.
   4. Incubate on ice for 30 min.
   5. Centrifuge at 10,000 x g in a pre-chilled centrifuge/rotor (4°C) for 10-15 min.
   6. Collect the supernatant from the cell lysate and transfer to a new tube.

      *see recipe at end of protocol.

3. Preclear Lysate
   1. Preclear the lysate by incubating with a control antibody (10 mg/ml for a monoclonal antibody; 1-5 ml for polyclonal antibodies) for 1 hr at 4°C with agitation. NOTE: Control antibody should be the same isotype as the primary monoclonal antibody; polyclonal antisera should be from the same animal (pre-immunization) or same species as the primary, specific antibody used.
   2. To 1 ml of cell lysate: Add 500 ml Protein G Sepharose^® or Protein A Sepharose^® bead slurry (10% v/v in lysis buffer or RIPA buffer containing protease inhibitors)
   3. Centrifuge at 10,000 x g for 15 sec at 4°C. Collect and aliquot the supernatant for precipitation with specific antibodies.
4. Incubation with Specific Antibody
   1. Using approximately 50 mg of total cellular protein, (e.g., lysate from ~2 x 10⁵ cells), add 1-5 mg of antibody per reaction.
   2. Incubate with rotation or agitation at 4°C for 2 hr.
5. Immunoprecipitation
   1. Add an equal volume of Protein G Sepharose^® or Protein A Sepharose^® bead slurry to cell lysate. Incubate with rotation or agitation at 4°C for an additional 1-2 hr. NOTE: The choice of Sepharose^® used is dependent on the isotype of the primary antibody to be used and the species in which it was made (see table 3)
   2. Centrifuge at 10,000 x g for 15 sec at 4°C.
   3. Aspirate the supernatant. Alternatively, the supernatant containing radiolabeled proteins may be transferred to a separate tube for immunoprecipitation with an additional/separate-specificity antibody.
6. Wash
   1. Wash the beads 3-4 times in cold lysis buffer containing 0.5 M NaCl. Use RIPA Buffer for a more stringent wash.
   2. Transfer the beads to a fresh tube and wash once more with cold lysis buffer.
   3. After the last wash, add 50 ml of 1X SDS-PAGE sample buffer* to beads. Elute the bound immunocomplex by boiling the beads for 2 min.

   4. Centrifuge at 10,000 x g for 1 min before loading supernatant on gel.

      *see recipe at end of protocol

7. Detection
   1. Resolve by SDS-PAGE.
   2. Stain gel with coomassie blue or silver stain, destain.
   3. Dry gel.
   4. Expose to X-ray film.