α mouse igg hrp

The cells cultured as described above were centrifuged and washed twice with cold PBS followed by freezing using liquid nitrogen. The frozen cells were suspended with lysis buffer (50 mM Tris–HCl (pH 7.5), 1 mM EDTA (pH 8.0), 10% glycerol, 150 mM NaCl, 0.05% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, Complete protease inhibitor (Roche)) and cells were disrupted by 0.5 mm zirconia beads and Multibead Shocker (Yasuikikai, Japan). The supernatants were then collected by centrifugation and the protein concentration was measured. Equal amounts of SDS buffer were added to the solutions and heated at 100°C for 3 min. The resulting samples were resolved by standard SDS-PAGE on a 12% polyacrylamide gel and transferred to a PVDF membrane by Wet transfer system. α-Atf1 antibody (ab18123, abcam), α-HA antibody (12CA5, Sigma), and α-tubulin antibody (T6074, Sigma) were used as primary antibodies, and α-mouse IgG-HRP (GE Healthcare) and α-rat IgG-HRP (Santa Cruz) were used as the secondary antibody for protein detection.

The cell extracts obtained by the procedure above were incubated with α-HA antibody (12CA5, Sigma) for 1.5 h at 4°C. This solution was then mixed with Dynabeads Protein A (Invitrogen), followed by incubation for 1.5 h at 4°C. Thereafter, the beads were washed 3 times with lysis buffer. 25 μl of TEV buffer (50 mM Tris–HCl (pH 8.0), 5 mM EDTA (pH 8.0), 1mM DTT) were added to the beads with or without adding 15 U TEV protease (T4455, Sigma) followed by overnight incubation at 30°C. α-HA antibody (12CA5, Sigma) was used as the primary antibody and α-mouse IgG-HRP (GE Healthcare) was used as the secondary antibody for protein detection.