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).
Protran premium nc membranes
Manufactured by GE Healthcare
Protran Premium NC membranes are made of high-quality nitrocellulose material designed for use in various laboratory applications. They provide a reliable and consistent platform for diverse protein-based assays and analyses.
Automatically generated - may contain errors
2 protocols using protran premium nc membranes
1
Worm Ubiquitylation Assay Protocol
2
Western Blot Analysis of Proteins
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Equal amounts of proteins or beads were resuspended in 2× SDS loading buffer (100 mM Tris–HCl [pH 6.8], 4% [w/v] SDS, 200 mM DTT and 20% [v/v] glycerol) and then heated at 95°C for 10 min. Samples were resolved onto an 8% SDS-PAGE and transferred to Protran Premium NC membranes (GE Healthcare). The membranes were blocked with milk (5% [w/v] dried milk in 1× PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.1% [w/v] Tween-20, [pH 7.2]) and later, incubated overnight at 4°C with primary antibodies. Rabbit polyclonal anti-ALG-1 and anti-AIN-1 antibodies were used at 1:1000 and 1:2000 dilution, respectively, in 1% [w/v] dried milk-supplemented PBST. Monoclonal mouse anti-beta-ACTIN (Abcam, catalogue #ab49900) antibody was used at 1:20 000 dilution in 1% [w/v] dried milk supplemented PBST. Anti-FLAG (Millipore Sigma, catalogue #F1804) antibody was used at 1:10 000 dilution in 1% [w/v] bovine serum albumin supplemented PBST. Membranes were then incubated with appropriate secondary antibodies before developing with Perkin-Elmer ECL Western Lightning Plus solution and visualized using a Chemidoc imaging system (Bio-Rad).
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