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Star Republic: Guide for Biologists

Immunoprecipitation

1. Immunoprecipitation (IP) of labeled cells is frequently followed by SDS-PAGE and dried-gel is exposed to a X-ray film. IP of un-labeled cells is usually followed by Western or an activity assay (e.g., kinase assay).

2. Wash cells twice with cold PBS.

3. Harvest cells (e.g., 35 mm plate for labeled cells) into 1 ml of the lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, plus proteinase inhibitor cocktail). Triton X-100 can be used in place of NP-40. 10 mM phosphate buffer (pH 7.2-7.5) is frequently used instead of Tris-buffer.

4. Freeze and thaw three times.

5. Centrifuge at maximum speed for 10 minutes at 4 C.

6. Transfer the supernatant to a fresh tube.

7. Preclear by adding 50 ml of agarose>protein A-agarose or protein G-agarose. Incubate at 4 C for 2-4 hours with rocking.

8. Pellet beads by breif centrifugation.

9. Transfer the supernatant to a fresh tube.

10. Add 1-2 ml of antibody or serum ( 20-50 ml for hybridoma supernatant). Incubate at 4 C for 1 hours with rocking.

11. Add 50 ml of protein A- or G-agarose. Incubate at 4 C for 2-4 hours or overnight with rocking.

12. Pellet beads by breif centrifugation.

13. Remove supernatant. Wash with 1 ml of of washing buffer (50 mM Tris-HCl pH7.5, 500 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate ) or PBS/1% NP-40.

14. Repeat wash step 12-13 for two times.

15. Proceed to follow up analysis. For SDS-PAGE, remove as much supernatant as possible and boil the protein/beads for 2 minutes in 30 ml gel loading buffer.