Western Blot of PLBD2
Western blot was performed to confirm the existence of PLBD2. Samples were prepared by mixing 12.5 µL mAb-1 (4 mg/mL) with 2.5 µL 0.25 M IAM and 10 µL 2x Tris-Glycine loading buffer, followed by heating at 80°C for 2 min. 20 µL sample was loaded onto the SDS-PAGE gel for electrophoresis separation at 160V for 1.5 hrs, and the separated proteins were transferred to PVDF membrane at 25V for 30min. The PVDF membrane was then blotted by 2% BSA in PBST for 1 hr at room temperature, followed by adding anti-PLBD2 monoclonal antibody in 1% BSA (1:1000) and incubating at 4°C overnight. After washing with PBST three times, the secondary antibody anti-goat IgG was added at 1:5000 at room temperature for 1 hr. The PVDF membrane was then washed by PBST three times and stained by 1-step ultra TMD-blotting solution.