Western lightening ecl kit

Hand-picked animals were harvested at the young adult stage. The worms were treated by rotating them in suspension for two hours with vehicle only (DMSO) or with 50μM of MG132 (Sigma-Aldrich, 474790) in M9. Harvested animals were collected and washed in M9 before being lyzed in SDS loading buffer (1 mM Tris-HCl [pH 6.8], 2% [w/v] SDS, 100mM DDT and 10% [v/v] glycerol). The homogenized extract was clarified by centrifugation at 17,000× g for 5 min at 4°C. To detect ALG-1or ACTIN, the total protein extract was boiled for 10 minutes in SDS loading buffer and proteins were resolved on 8% acrylamide gel and transferred to Protran Premium NC membranes (GE Healthcare). Membranes were incubated overnight at 4°C with either antibody: (i) Rabbit polyclonal against ALG-1 diluted 1:1,000 or (ii) Mouse monoclonal against beta-ACTIN (Abcam, ab49900) diluted 1:20,000; Ubiquitin (Santa Cruz, sc-8017) diluted 1:400. Antibodies were diluted in PBST-1% bovine serum albumin solution (137mM NaCl, 10 mM Phosphate, 2.7mM KCl [pH 7.4], 0.05% [v/v] Tween-20 and 1% [w/v] bovine serum albumin). The membrane was incubated for 1 hour at room temperature with HRP-conjugated secondary antibody in PBST and then revealed using Western Lightening ECL Kit (Perkin Elmer) and visualized using Chemidoc imaging system (BioRad).