Immunoprecipitation
Kirk Lab Protocol
Immunoprecipitation - Mass Spec
Magnetic Bead Prep
1. Briefly vortex the stock tube to resuspend magnetic beads
2. Wash beads 2 times using 200 ul TBS-T and magnetic separation rack
3. Proceed to step 3 below
Immunoprecipitation
1. Add primary antibody (at appropriate dilution – usually 2-3 ug) to 300 ul of cell lysate (should contain 400+ ug of protein; it is okay to dilute sample to appropriate volume using TBS-T)
2. Incubate overnight with rotation at 4° C to form the immunocomplex
3. Transfer the lysate + antibody solution to the tube containing the pre-washed magnetic bead pellet
4. Incubate with rotation for 20 minutes at room temperature
5. Briefly centrifuge and then pellet beads using magnetic separation rack.
6. Save 20 ul of flow through (supernatant) for later. Discard rest.
7. Wash beads 4-5 times using 200 ul of TBS-T. Keep on ice between washes
8. Resuspend bead pellet in 100 ul of TBS-T and transfer to new Eppendorf tube.
9. Eluting the sample:
- a. Place tube on magnetic separation rack and remove supernatant
- b. Add 30 ul SDS-Tris Glycine supplemented with 5 ul Bolt reducing buffer to beads and resuspend
- c. Boil at 100° C for 10 minutes.
10. Pellet beads using magnetic separation rack. Transfer supernatant to a new tube (THIS IS YOUR SAMPLE).
11. Load sample (35 ul) on SDS-PAGE gel
12. Run gel at 80 mV for 10 minutes and then 170 mV until complete. Fix for 15 minutes. Stain with Coomassie Blue for 3-6 hours.
13. Destain overnight. Move to Mass Spec prep protocol.
