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.