AFFINITY PURIFICATION OF PROTEIN COMPLEXES

Lysis/IP buffer

150, 300, or 500 mM NaCl; 0.1% NP-40; 20 mM Tris-HCl, Ph7.8; 0.5 mM DTT; 0.5 mM EDTA; 10 mM NaF; 0.3 mM Na3VO4; 10 mM b-Glycerolphosphate; 10 ug/ml Aprotonin; 1 ug/ml Leupeptin; 1 ug/ml Pepstatin A; 1 mM PMSF

Procedure:

• Spin down the cells in the bench-top centrifuge at 1500 rpm for 10 minutes at 4 C.
• Wash cell pelltes twice with PBS (Cold).
• Resuspend cell pellet in X ml lysis/IP buffer (cold, be sure add protease inhibitors). About 3-5 ml of lysis/IP buffer per litter of cells, or 10 mg/ml final protein extracts
• Transfer cells into a 50 ml tube.
• leave the tube on ice for 30 minute with shaking every 5 minutes.
• Spin cell lysate in a centrifuge at 2K rpm for 5 minutes
• Transfer the supernatants into a 50 ml ultracentrifuge tube and spin for 30 minute at 60K.
• Incubate supernatant with antibody-conjugated beads at 4 C for 3-6 hrs. 5-10 ul beads per ml of cell extracts. Total volume of beads should be less than 200 ul.
• Wash with 1 ml of the same lysis/IP buffer for five times with a few minutes rotations each time.
• Wash once with 100 mM NaCl IP buffer
• Incubate with 1-2 bed volume of peptide elution buffer (100 mM NaCl, 0.1% NP40, 0.2 mg/ml peptide) for 30 minutes at 4 C.
• Apply the elution mixture to compact reaction column and put the column onto 1.5 ml Eppendorf tube and spin.
• Repeat the elution process for 2 times.
• Run SDS-PAGE gel using Tris-Glycine buffer, and include four additional lanes (in addition to your sample lane) in your SDS-PAGE gel: molecular weight markers, experiment control, a 1 picomole BSA control, and a 5 picomole BSA control.
• Visualize the protein with the Colloidal Blue Staining method (Novex, San Diego, CA).
• Indicate the protein bands to be analyzed in a gel picture.
• Wrap the gel with Saran wrap plastic film and send it to Mass-spec laboratory in a lunch box with a water-ice brick by express mail. Please do not cut out the protein bands. The sample history (purification, information about the project, etc) also must accompany each shipment.