Polyscreen pvdf membrane

Cells were grown to mid-log phase, pelleted and washed with dH₂O, and frozen in liquid nitrogen. Pellets were resuspended in 350 μl IPH150 or IPH50 buffer (50 mM Tris, pH 8.0, 150/50 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM DTT, protease inhibitors cocktail (Sigma)). Cells were lysed by adding glass beads (Sigma) to the re-suspended pellets followed by 4 working cycles of 1 minute in a bullet blender (Next Advance). The lysates were cleared by two centrifugations of 5 and 15 min at 15,000 g at 4°C. Immunoprecipitations were performed at 4°C adding the appropriate antibodies for 2 h. The antibodies were collected on protein A/G agarose (Santa Cruz) for 1 h and washed 3 times with IPH150 and resuspended in 35 μl Laemmli buffer. Standard procedures for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting were followed to transfer proteins from gels to a polyscreen PVDF membrane (Millipore). Membranes were blotted with the primary antibodies. Antibodies were detected using SuperSignal West Pico (Thermo) and LAS 4000 (GE). Antibodies used in this study: anti-HA (12CA5, Roche), anti-MYC (9E10, Roche), anti-V5 (Invitrogen/Millipore), Rabbit anti-Mcd1 (Rb555) and Rabbit anti-Pds5 (Rb558) antibodies were provided by Vincent Guacci.

Cells were grown to midlog phase, pelleted, washed with dH₂O, and frozen in liquid nitrogen. Pellets were resuspended in 350 μl IPH50 [50 mM Tris pH 8.0, 50 mM NaCl, 5 mM EDTA, 0.5% NP-40, 5 mM β-mercaptoethanol, protease inhibitor cocktail (Sigma)]. Cells were lysed by adding glass beads (Sigma) to the resuspended pellets followed by four working cycles of 1 min in a bullet blender (Next Advance). The lysates were cleared by two centrifugations of 5 min and 15 min at 1000 × g and 14,000 × g, respectively, at 4°. Immunoprecipitations were performed at 4° adding the appropriate antibodies for 2 h. The antibodies were collected on protein A/G agarose (Santa Cruz) or on magnetic beads (BIO-RAD) for 1 hr, and washed three times with IPH50 and resuspended in 35 μl Laemmli buffer. Standard procedures for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting were followed to transfer proteins from gels to a polyscreen PVDF membrane (Millipore). Membranes were blotted with the primary antibodies. Antibodies were detected using SuperSignal West Pico (Thermo Scientific) and LAS 4000 (GE Healthcare). Antibodies used in this study were: anti-HA (12CA5, Roche), anti-MYC (9E10, Roche), anti-V5 (Invitrogen/Millipore), anti-3Flag (Sigma), and anti-6His (Sigma).