Mouse monoclonal anti flag m2

For immunoblots of C. elegans lysate, ∼60 adult hermaphrodites were collected into 500 μl M9 buffer and washed 2 × with M9 buffer and 2 × with M9 buffer containing 0.05% Triton X-100. To 100 μl of worm suspension, 33 μl 4× SDS-PAGE sample buffer (250 mM Tris-HCl, pH 6.8, 30% [v/v] glycerol, 8% [w/v] SDS, 200 mM DTT and 0.04% [w/v] bromophenol blue) and ∼20 μl of glass beads were added. Samples were incubated for 3 min at 95°C and vortexed for 2 × 5 min with intermittent heating. After centrifugation at 20,000 × g for 1 min at room temperature, proteins in the supernatant were resolved by 10% SDS-PAGE and transferred to a 0.2-μm nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech). Membranes were rinsed 3 × with TBS (50 mM Tris-HCl, 145 mM NaCl, pH 7.6), blocked with 5% non-fat dry milk in TBST (TBS containing 0.1% Tween 20), and probed at 4°C overnight with mouse monoclonal anti-FLAG M2 (F3165, 1:1,000; Sigma-Aldrich) and mouse monoclonal anti-α-tubulin B512 (T5168, 1:5,000; Sigma-Aldrich). The membrane was washed 3× with TBST, incubated with goat polyclonal anti-mouse IgG antibody coupled to HRP (115-035-044, 1:10,000; Jackson ImmunoResearch) for 1 h at room temperature, and washed again 3× with TBST. Proteins were detected by chemiluminescence using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and x-ray film (GE Healthcare).