Establishment of minimal positive-control conditions to ensure brain safety during rapid development of emergency vaccines

Baek, Hyekyung; Kim, Kwang Ho; Park, Min Young; Kim, Kyeongryun; Ko, Bokyeong; Seo, Hyung Seok; Kim, Byoung Soo; Hahn, Tae-Wook; Yi, Sun Shin

Protein concentrations were determined by using a BCA kit (iNtRon Biotechnology). Total proteins (20 g per sample) were loaded to each lane of 12% SDS-PAGE, electrophoresed, and transferred to PVDF membranes (Bio-Rad Laboratories, USA). Following transfer, membranes were blocked with TBST [100 mM Tris-HCl (pH 7.6), 0.8% NaCl, and 0.1% Tween-20] containing 5% skim milk (BD Biosciences, USA). The blocked membranes were incubated with diluted rabbit anti-ZO-1 (1:2,000; Thermo Scientific, USA), mouse anti-occludin (1:2,000; Thermo Scientific), and rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, USA) primary antibodies at 4 o C overnight. After further washing, membranes were incubated with peroxidase-labeled anti-rabbit and anti-mouse secondary antibodies (Vector, USA). Immunoreactive signals were detected by using an enhanced chemiluminescence reagent (Abclon, Korea) and recorded by using a MicroChemi 4.2 system (DNR Bio-Imaging Systems, Israel).