G sepharose beads

Drosophila S2 cells were cultured in M3 media (Sigma) with 10% Insect medium supplement (Sigma). Transfection was carried out with Effectene reagent (Qiagen) according to manufacturer’s instructions. Total of 1–2 μg DNA was used for each transfection. For immunoprecipitation, cells were lysed in 0.1% CHAPS buffer, and the lysates were precleared by incubating with protein G-sepharose beads (Roche) for 1 h at 4 °C. The G-sepharose beads were immunoprecipitated with anti-Myc (Abcam) or anti-Flag (Sigma) at 4 °C for 1 h. The immunoprecipitates captured by protein G-sepharose were incubated with the clear lysates overnight at 4 °C. Immunoprecipitates were washed three times in cold IP buffer. The samples were boiled in protein loading buffer at 94 °C for 5 min and then subjected to SDS-PAGE. Western blot was performed with mouse Flag (Sigma) or mouse V5 at 1:5000 (Invitrogen).

For Western analysis, samples were mixed with 4 × SDS sample buffer and boiled at 95 °C for 10 min. Samples were then separated by 10–12% SDS-PAGE and transferred to the nitrocellulose membrane (Millipore). Membranes were blocked with 5% nonfat milk in TBST buffer (10 mM Tris pH 7.4, 0.8% NaCl, 0.1% Tween-20), and probed with the antibody. After washing membranes with TBST several times, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody in TBST with 5% nonfat milk. After washing, protein bands were visualized using the ECL system (AbFrontier).
For co-immunoprecipitation, cells were lysed in HEPES buffer (20 mM HEPES, 70 mM KCl, 2 mM DTT, 0.1% NP40, 8% Glycerol, 1 mM PMSF, 10 mM EDTA, 10 mM EGTA, and protease inhibitor cocktail (Roche)) on ice. The lysates were precleared by incubating with protein G-sepharose beads (Amersham Bioscience) for 30 min at 4 °C. A new set of G-sepharose beads were incubated with anti-GFP (Abcam, rabbit) or Sona-Pro (rabbit) for coupling at room temperature for 2 hrs. The precleared lysates were then incubated with coupled protein G-sepharose beads for overnight at 4 °C. The protein G-sepharose beads were washed with HEPES buffer and Western blots were performed as described.