Immunoprecipitation of RASSLs

1. Add the following to the supernatant of the solubilized sample:

• 2 ml of 1 M CaCl₂
• 2 ml of M1-Antibody (1mg/µl) (Eastman Kodak Co. #IB13007)
• 15 ml of 50% suspension Prot-A agarose beads (Sigma # P-2545)

*IP Standard, use 50 ng of a flag-tagged protein.

2. Incubate overnight at 4°C on a rotator.

3. Spin down beads for 1 min at 4°C at 3,000 rpm.

4. Discard supernatant using a pipet or a vacuum. Be careful not to remove beads.

5. Add 1 ml of TBS-T Ca⁺⁺ Wash buffer to beads. Invert tubes a few times and spin for 1 min at room temperature at 3,000 rpm.

6. Discard supernatant using a pipet or a vacuum.

7. Repeat wash (steps No. 5 and No. 6).

8. Add 50 ml of 1x electrophoresis sample buffer to beads.

9. Vortex. Boil sample for 5 minutes.

10. Vortex. Spin for 2 min at room temperature at 5,000 rpm.

11. Transfer supernatant to new tube using flat tips or load supernatant on gel using flat tips. Pipet carefully and try not to disturb bead pellet.

Buffers

• 1x TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl)
• 0.1% Tween-20
• 1 mM CaCl₂

Sample Buffer:

• 2x Tris-Glycine SDS Sample Buffer (Novex #LC2676) plus 5% beta-mercaptoethanol dilute to 1x with ddH₂0

Back to Conklin Lab home page