Peroxidase conjugated rabbit anti mouse igg secondary antibody

RD cells grown in a 24-well plate were transfected and infected as described above. To obtain sufficient viral protein, each experimental group was treated in two independent wells. At 36 h postinfection, the cells were harvested and lysed with RIPA lysis buffer (Beyotime) to determine the protein concentrations. Equal amounts of protein were separated electrophoretically in an SDS-polyacrylamide gel (12%), and western blotting was performed with standard procedures. The viral protein was detected with a mouse anti-EV71 VP1 monoclonal antibody (Abcam, USA) at a dilution of 1: 1000, and a mouse anti-β-actin antibody (Abcam) at a dilution of 1: 1000 was used as the internal control. A peroxidase-conjugated rabbit anti-mouse IgG secondary antibody (Abcam, USA) was used at a dilution of 1: 5000. The bound antibody was visualized with ECL chemiluminescent substrate (Beyotime).

Overnight cultures of transformed L. plantarum WCFS1 were diluted 1:100 into fresh MRS containing 5 μg/mL of erythromycin, twice. An amount of 1% (v/v) of the above transformed L. plantarum WCFS1 was transferred into 10 mL fresh MRS medium containing 5 μg/mL of erythromycin and incubated statically at 37 °C for 12 h. Then the bacteria and supernatant were separated by centrifugation. The supernatant was precipitated with trichloroacetic acid (10% w/v) for 1 h at 4 °C, and the mixture was then centrifuged at 12,000 rpm for 10 min. The precipitate was washed three times with ice-cold acetone and finally dissolved in 100 μL PBS.
The concentrated culture supernatant was mixed with 5× SDS loading buffer and subjected to western blot analysis in a 16.5% Tricine-SDS-PAGE gel. Proteins were then semi-dry-electrotransferred onto a PVDF membrane, and binding was performed sequentially using the mouse 6× His antibody (Abcam, Cambridge, UK) and the peroxidase-conjugated rabbit-anti-mouse IgG secondary antibody (Abcam, Cambridge, UK). After washing three times, the membrane was subjected to ECL chemiluminescent kit (ShareBio, Shanghai, China) and analyzed. As for the enzyme-linked immunosorbent assay (ELISA), 10 μL of culture supernatant was sampled every 3 h of GLP-1 secretion and quantified by His-tag ELISA kit (Sangon Biotech, Shanghai, China) according to manufacturer’s instructions.