Horseradish peroxidase conjugated secondary antibodies mouse and rabbit

Approximately ten A₆₀₀ cells (∼10 ml of cells at A₆₀₀ ∼1) were collected from the respective cultures, pelleted, and flash-frozen in liquid nitrogen until further use. The cells were resuspended in 400 μl of 10% TCA and lysed by bead beating three times: 30 s of beating and then 1 min of cooling on ice. The precipitates were collected by centrifugation, resuspended in 400 μl of SDS-glycerol buffer (7.3% SDS, 29.1% glycerol and 83.3 mm Tris base), and heated at 100 °C for 10 min. The supernatant after centrifugation was treated as the crude total protein extract. Protein concentrations from extracts were estimated using a bicinchoninic acid assay (Thermo Scientific). Equal protein amounts of each sample was resolved on 4–12% Bis-Tris gels (Invitrogen). Coomassie Blue–stained gels were used as loading controls. Western blots were developed using antibodies against the respective tags. The following primary antibodies were used: monoclonal FLAG M2 (F3165, Sigma), HA (12CA5, Roche), and phosphor-eIF2α Ser51 (9721, Cell Signaling). Horseradish peroxidase–conjugated secondary antibodies (mouse and rabbit) were obtained from Sigma. The molecular weight markers used were 2661 (Thermo Scientific) and PG-P MT2922 (Genetix). For Western blotting, standard enhanced chemiluminescence reagents (GE Healthcare) were used. ImageJ was used for quantification.