Western blot analysis was performed on Membrane extracts (30 µg lysate) of K-562 (Lane 1), K-562 treated with H2O2 (150 uM for 1 hr) (Lane 2), HEL 92.1.7 (Lane 3), HEL 92.1.7 treated with H2O2 (150 uM for 1 hr) (Lane 4), T-47D (Lane 5), T-47D treated with H2O2 (150 uM for 1 hr) (Lane 6), HEK-293 (Lane 7) and HEK-293 treated with H2O2 (150 uM for 1 hr) (Lane 8). The blots were probed with Anti-eIF2AK1/Heme regulated inhibitor (HRI) Recombinant Rabbit Monoclonal Antibody (Product # 702551, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 71 kDa band corresponding to eIF2AK1/Heme regulated inhibitor (HRI) was observed across the cell lines (with treatment difference) tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).
Western blot analysis was performed on Membrane extracts (30 µg lysate) of K-562 (Lane 1), K-562 treated with H2O2 (150 uM for 1 hr) (Lane 2), HEL 92.1.7 (Lane 3), HEL 92.1.7 treated with H2O2 (150 uM for 1 hr) (Lane 4), T-47D (Lane 5), T-47D treated with H2O2 (150 uM for 1 hr) (Lane 6), HEK-293 (Lane 7) and HEK-293 treated with H2O2 (150 uM for 1 hr) (Lane 8). The blots were probed with Anti-eIF2AK1/Heme regulated inhibitor (HRI) Recombinant Rabbit Monoclonal Antibody (Product # 702551, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 71 kDa band corresponding to eIF2AK1/Heme regulated inhibitor (HRI) was observed across the cell lines (with treatment difference) tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).
Supplier Page from Thermo Fisher Scientific for HRI Antibody