Mouse anti 6 his antibody

Antigen was separated by 12% SDS-PAGE. It was stained by CBB dye for direct visualization of the protein band(s) or its separated components were electroblotted onto a nitrocellulose membrane (NC; Bio-Rad) for Western blot analysis. For the latter, the empty sites on the blotted NC were blocked with unrelated protein (3% skim milk or BSA in PBS), and the membrane was cut vertically into strips. The NC strips were probed individually with primary antibody, i.e., mouse anti-6×-His antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) to trace the SDS-PAGE-separated rPer a 1.0105 band or hybridoma culture supernatants and to check for the presence/antigenic specificity of the mouse monoclonal antibody to rPer a 1.0105. In both instances, goat anti-mouse IgG-alkaline phosphatase (AP) conjugate and BCIP/NBT chromogenic substrate (KPL, Gaithersburg, MD, USA) were used as reagents to reveal the antigen-antibody reactive band(s).

The cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes. The membranes were then blocked with Tris-buffered saline and Tween-20 contaning 5% of bovine serum albumin at room temperature for 2 h and incubated with corresponding antibodies overnight at 4 °C. The membranes were washed and incubated with appropriate dilution of IRDye 800 donkey anti-mouse IgG or IRDye 700 donkey anti-rabbit IgG (LI-COR Inc., Lincoln, NA, USA) for 1 h at room temperature. After washing with Tris-buffered saline, the membranes were then analyzed by an Odyssey Infrared Imaging System (LI-COR Inc.). Rabbit anti-poly (ADP-ribose) polymerase antibody, goat anti-CAR antibody, mouse anti-6-his antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Mouse anti-E1A antibody was purchased from Abcam (Cambridge, UK). Goat anti-hIL-3 antibody was purchased from R&D Systems. Rabbit anti-MnSOD antibody was purchased from Epitomics (Burlingame, CA, USA). Rabbit anti-GAPDH antibody, rabbit anti-Bcl-2 antibody, rabbit anti-Bax antibody, rabbit anti-caspase-7 antibody and rabbit anti-caspase-9 antibody were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).