Horseradish peroxidase hrp conjugated igg

Spinal cords were dissected and homogenized in 10 volumes of cold RIPA buffer (Tris-HCl pH 7.5, 50 mM; NaCl, 150 mM; NP-40, 1%; sodium deoxycholate, 0.5%; SDS 0.2%) supplemented with complete protease inhibitors (Roche). Homogenates were then incubated on ice for 30 min and spun for 10 min at 16000×g to remove cell debris. Supernatants were collected and total proteins were quantified using the Bicinchoninic (BCA) Protein Assay Kit (Pierce). About 40 μg of total proteins were separated by SDS-PAGE on 10% polyacrylamide gels and then blotted onto nitrocellulose membranes (Immobilion) at 100V for 30 min. Membranes were subsequently blocked with 5% milk in Tris buffered saline with 0.05% Tween-20 (TBS-T) for 1 hour at room temperature (RT). After blocking, membranes were incubated with primary antibodies diluted (1:1000) in blocking solution or 5% bovine serum albumin (BSA) in TBS-T overnight at 4°C. The day after, the membranes were washed 3 times with TBS-T and incubated with horseradish peroxidase (HRP)-conjugated IgG (Cell Signal Technology) in blocking solution (1:5000) for 1 hour at RT. After extensive washing, membranes were incubated with Supersignal West Dura reagent (Thermo Scientific) and the chemiluminescent signals were detected using a Molecular Imager ChemiDoc XRS System equipped with Quantity One software (Bio-Rad).