Anti his tag mouse monoclonal antibody

After the centrifugation of the disrupted CeNVD_pET-28a(+), the cleared supernatant and precipitant were loaded on SDS–PAGE gels, then all the protein molecules was transferred to a PVDF membrane and blocked in a PBST buffer (PBS pH 8.0, 0.02% Tween-20) containing a 1% bovine serum albumin (BSA) for 2 h, followed by incubation in an anti-His-tag mouse monoclonal antibody (Abcam, Cambridge, United Kingdom), which was diluted in a blocking buffer (PBST pH 8.0, 1% BSA) at the indicated concentrations of 1:5,000 for 12 h at 4°C. After washing with PBST for four times, the membrane was protected from light and incubated with the HRP-conjugated secondary antibody (HRP-conjugated goat anti-mouse IgG, Tiangen Biochemical Technology, Beijing, China) at a dilution of 1:1,000 and room temperature for 2 h. After washing, the target protein was trapped by the HRP-DAB chromogenic substrate kit (Tiangen Biochemical Technology, Beijing, China), and the immunoreactive band was digitally scanned using an Odyssey Infrared Imager (LI-COR Bio-science, Lincoln, NE, United States) (Wan et al., 2016 (link)).

The recombinant protein was mixed with loading buffer, boiled at 100 °C for 5 min, centrifuged at 12,000× g for 1 min, and separated by 12% SDS-PAGE gel before being transferred to a PVDF membrane (Burlington, MA, USA) by a trans-blot SD semi-dry electrophoretic transfer cell (Beijing Liuyi, China). Then, the PVDF membrane was incubated with 5% BSA for 2 h, primary antibody (Anti-His Tag Mouse Monoclonal Antibody, Abcam, Cambridge, UK) overnight, and secondary antibody (HRP Goat Anti-Mouse lgG, Abcam, Cambridge, UK) for 2 h. Each time the incubation solution was changed, 1× TBST was used to wash the PVDF membrane. Finally, the target band on the PVDF membrane was dyed by using the Pierce™ ECL Plus Western blotting substrate (ThermoFisher, Waltham, MA, USA) and detected by using the chemiluminescent imaging system.