Anti 6xhis monoclonal antibody

Echelon Biosciences PIP Strips were incubated with 80 nM rABH proteins in PBS containing 0.25% Tween (PBST) and 2% powdered skimmed milk (SM-PBST) overnight at 4 °C followed by three washes in 1 × -PBST buffer. rABH was detected using anti-6xHis monoclonal antibody (1:5,000, Sigma, #H1029) followed by anti-mouse IgG-horseradish peroxidase (HRP) conjugated antibody (1:10,000, Jackson Laboratories, #111-035-003). The blot was developed using chemiluminescence substrate solution (Pierce) and X-ray film.

Samples from the E. coli culture, which had been transformed by recombinant plasmids, were added to the sodium dodecyl sulfate (SDS) buffer and beta-mercaptoethanol. Then, after heating at 100°C for 10 min, the samples were electrophoresed on 12% polyacrylamide gel. The proteins were then electroblotted onto a nitrocellulose membrane (GE Healthcare Life Sciences). The membrane was blocked with 5% skimmed milk in PBS (1h/37°C) and then washed with PBS 0.05% Tween (PBS-T) (3 times for 5 min each), before being incubated with the anti-6X-his monoclonal antibody 1 : 4000 (Sigma) (1h/37°C). After three membrane washes with PBS-T, peroxidase-conjugated anti-mouse IgG antibody (Sigma), diluted in PBS-T (1 : 4000), was added. Finally, the incubation process (1h/37°C) was performed before revealing it with 3,3′-diaminobenzidine (DAB) and H₂O₂.

Anti-c-myc monoclonal antibody (Clontech, USA; 1:1,000), anti-GAPDH antibody (Cell Signaling Technology, USA; 1:10,000), anti-6X-His monoclonal antibody (Sigma; 1:2,000), anti-p24 monoclonal antibody (NIH AIDS Reagent Program, USA; 1:3,000), anti-PARP(Cell Signaling Technology, USA; 1:1000), anti-Rabbit IgG conjugated to HRP (Jackson Immunoresearch, USA; 1:10,000) and anti-Mouse IgG conjugated to HRP (Jackson Immunoresearch; 1:10,000) were used for the western blotting experiments.