Immunoprecipitation Protocol Using Simple Western

1. Cell lysis

Lysis Buffer: 50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1.1% octyl glucoside.

1. Lyse cells in buffer.
   • Note: All cells are different in size and protein concentration. Protein concentration should be approximately 5 mg/mL. Clear lysate by centrifugation at 15,000 rpm for 10 minutes at 4 °C.
2. Estimate protein by BCA assay (Pierce, #23225)

2. Immunoprecipitation protocol

Using either a biotinylated antibody (when using SA beads) or biotinylated peptide:

1. Mix 2 mg of cleared protein lysate with either 100 µM peptide or 100 µg/mL antibody, overnight at 4 °C, continuous rocking.
2. After 24 hours, prepare streptavidin magnetic beads.
   1. Briefly take 50 µL of beads Streptavidin magnetic beads (Pierce: cat#88817) and wash three times with excess PBS (1.5 mL each time).
3. Add immunoprecipitation complex to cleared beads. Rock for approximately 1 hour at room temperature.
4. Using a magnetic column, isolate beads and remove lysate (retain for future use if desired). Wash three times with excess lysis buffer.

3. Prepare samples for Peggy Sue analysis

1. Add 65 μL of sample standard in 1x MM format directly to the beads.
2. Heat at 9 5°C for 5 minutes to elute protein from the beads.
3. Clear the beads using a magnet.
4. Retain 1x MM eluate solution containing the immunoprecipitation mixture.
5. Follow the instructions in the Product Insert of the Peggy Sue™ Separation Module (5 μL immunoprecipitation sample per well).