Purified anti-JNK Phospho (Thr183/Tyr185)

Brain tissue is homogenized with a glass-glass Potter apparatus in 0.32 M ice-cold sucrose buffer containing the following concentrations (in mM): 1 HEPES, 1 MgCl2, 1 EDTA, 1 NaHCO3, at pH 7.4, with a cocktail of protease and phosphatase inhibitors and stored at −80 °C until further analysis. Protein concentrations are quantified using the Bradford Assay and 10 μg of total homogenates are separated by 10% SDS polyacrylamide gel electrophoresis. PVDF membranes are blocked in Tris-buffered saline (5% BSA, 0.1% Tween 20), (1 h, room temperature). The primary antibody is diluted 1:1000 in the same buffer (incubation overnight, 4 °C). Blots are developed using a non-Abcam horseradish peroxidase-conjugated secondary antibody and the ECL chemiluminescence system (exposure 10s). The antibody is specific: there are two bands, at 54 and 46 kDa.

Review Date: 25 Apr 2020 | BioLegend