Anti sod1 antibody

WT and FAIM KO HeLa cells were transiently transfected with an eGFP-tagged native human SOD1 or aggregation-prone human SOD1-G93A protein expression vector, and fluorescently tagged cells were then harvested at 48 h. Cells were washed with PBS and then lysed in PBS containing 2% SDS, 1 mM MgCl₂, protease inhibitor cocktail and 25 unit/ml Benzonase (Merck). Protein concentrations were quantified using 660 nm Protein Assay Reagent with Ionic Detergent Compatibility Reagent (IDCR) (Thermo Scientific). Equal amounts of protein extracts underwent vacuum filtration through a 0.2 μm pore size cellulose acetate membrane (GE Healthcare) for the detection of SOD1 aggregates using a 96 well format Dot-Blot apparatus (Bio-Rad). The membrane was washed twice with 0.1% SDS in PBS and western blotted using anti-GFP antibody (Cell Signaling Technology) to detect aggregated proteins. Cell free aggregates of SOD1-G93A were similarly applied to nitrocellulose membrane. In cell-free experiments, SOD1 protein was vacuum filtered as above, after which membranes were western blotted using anti-SOD1 antibody (Cell Signaling Technology) to detect aggregated proteins.

Protein concentrations were determined using the 660 nm Protein Assay Reagent (Pierce). Protein samples in 1 × Laemmli buffer with 2-mercaptoethanol at 2.5% were boiled for 5 min. Equal amounts of protein for each condition were subjected to SDS-PAGE on an AnykD gradient gel (Bio-Rad) followed by immunoblotting with anti-SOD1 antibody (Cell Signaling) after wet transfer for 1 h to PVDF membrane (Bio-Rad) and blocking with non-fat dry milk.