Cell Surface Labeling Reagent From Thermo Fisher

Results Summary

1. Seed 1.5 million NIH-3T3 cells per 10-cm plate.2. 60 hr post seeding, confluent cells were serum starved for 24 h (25 nM SHH was included at this step).3. After 24 h, start cell surface biotinylation.4. Prepare a fresh solution of EZ-Link Sulfo-NHS-SS-Biotin in enriched PBS pH 7.4 (0.4 mM final conc.).5. Transfer plates to the cold (4 °C) room. 6.Quickly wash plates (thrice) with ice cold enriched PBS pH 7.4.7. Place plates on a metal rack chilled to 0 °C on ice and add 8 ml of biotinylation reagent/plate and incubate for 30 min.8.Add 425 µl of 1M Tris pH 8.0 (final conc. ~50 mM) per plate to quench any non-reacted biotinylation reagent and incubate for 10 min at 0 °C.9.Aspirate media and rinse plates thrice with 1xTBS pH 7.2 buffer.10. Add 1 ml of TBS pH 7.2 buffer (with 1x protease inhibitor cocktail) per plate and scrape cells into 1.5 ml tubes.11.Spin tubes at 400xg for 5 min and discard the supernatant.12. Add 500 µl lysis buffer directly on the plate (50 mM Tris-HCl pH-8.0, 150 mM NaCl, 2% NP-40, 0.25% Deoxycholate, 1x Sigma-Fast protease inhibitor cocktail, 1x Roche phosphatase inhibitor cocktail) and scrape lysates into a 1.5 ml eppendorf tube.13. Samples were incubated at 4 °C on a shaker for 45 min.14. Samples were spun at 20,000xg for 30 min at 4 °C.15. Wash 20 µl of Streptavidin agarose resin (Vector Laboratories) with lysis buffer (3x rapid washes).16. Set up streptavidin pull down with 600 µg lysate and 10 µl resin (in a 500 µl volume) for 2 h at 4 °C on a rotator.17. Wash beads five times with lysis buffer. 18. Elute proteins by adding 50 µl of 1x Laemmli buffer (containing 100 mM DTT) and incubating at 37 °C for 1 h.19. Run SDS-PAGE on 4-12% Bis-Tris gels with 5% inputs and 50% elutions and immunoblot for Smoothened.20. Good enrichment of Smoothened was observed.