Mouse anti gst antibody

Twelve percent SDS-polyacrylamide gel was normally used to analyze the recombinant proteins. SDS-PAGE was performed in the Mini-Protean system (Bio-Rad). After electrophoresis, the gels were either Coomassie Brilliant Blue R250-stained to visualize the protein bands, or transferred onto a nitrocellulose (NC) membrane (Schleicher & Schuell BioScience, Dassel, Germany). The membrane was blocked for 1 h at room temperature with 5% (w/v) skim milk in TBS-T buffer (140 mM NaCl, 2.7 mM KCl and 25 mM Tris, pH 7.4; containing 0.05% (v/v) Tween-20). The membrane was incubated in mouse anti-GST antibody (Sigma) diluted 1:1,000 in TBS-T containing 5% (w/v) skim milk for 2 h at room temperature, and washed three times (each time for 10 min) in TBS-T buffer, followed by incubation with peroxidase-conjugated goat anti-mouse IgG antibody (Capple, West Chester, PA) diluted 1:2,000 in 5% (w/v) skim milk. After washing, the ECL detection system (Amersham Biosciences) was added according to the manufacturer’s instruction for 1 min. Treated NC membrane was exposed to an X-ray film.