Anti gst tag mouse monoclonal antibody

The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells (Tiangen) and cultured in a shaker of 37 °C and 180 rpm. When the OD₆₀₀ reached about 0.6, IPTG was added (final concentration 1 mmol/l; Sigma-Aldrich, St. Louis, MO, USA) to induce and express recombinant vimentin protein. The cell pellet was collected by centrifugation and lysed with sonication at 4 °C. Then the lysed product was analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to confirm that the recombinant protein was present as a soluble protein or in inclusion bodies. The recombinant protein was purified according to the characteristics using a published gel purification method [18 (link)]. Purified recombinant vimentin protein (10 μg) was analyzed by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The PVDF membrane was blocked in 5% skim milk for 2 h at 37 °C and incubated with Anti-GST Tag Mouse Monoclonal Antibody (1:2000; CWBio, Beijing, China) for 2 h at 37 °C, and was then washed in phosphate-buffered saline (PBS) three times for 5 min and incubated with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (1:5000; Proteintech, Chicago, IL, USA) for 45 min at room temperature. Finally, imaging was obtained using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).

Proteins were separated by 10% SDS–PAGE and western blotting was performed with Anti GST-Tag Mouse Monoclonal Antibody (1000 × dilution, Cat No: CW0084M, CWBIOtech, Beijing, China)^{50 (link)}. Calibration curves were constructed using BSA (0.5 mg ml⁻¹) as standard in the range from 0 to 20 μl measuring absorbance at 595 nm. The purified NR protein was analyzed by applying standard Bradford assays at 595 nm. Nitrate Reductase (NR) activity was measured as described^{51 (link)}. Briefly, the reaction mixture contained 0.4 mL GST-fusion protein, 1.2 ml 0.1 mmol L⁻¹ potassium phosphate buffer (pH 7.5), 0.1 mmol L⁻¹ KNO₃, and 0.4 ml 0.25 mmol L⁻¹ nicotinamide adenine dinucleotide phosphate (NADPH). Controls substituted 0.1 mmol L⁻¹ potassium phosphate buffer (pH 7.5) for the NADPH. After 30 min incubation the NO₂⁻ produced was colorimetrically measured at 540 nm following addition of 10 g kg⁻¹ sulfanilamide in 2 mol L⁻¹ HCl and 0.2 g kg⁻¹ N-(1-naphthyl)-ethylene-diammonium dichloride (NED). NR activity was expressed as μg NO₂⁻ mg⁻¹ protein h⁻¹.