Transblot blot sd semi dry transfer cell

Proteins separated by SDS-PAGE were transferred onto PVDF membranes separately for both polyclonal and monospecific antibodies using a Transblot-Blot SD semi dry transfer cell (Bio-Rad) at 15 V for 2 hours (1x transfer buffer: Tris/Glycine with 20% Methanol). After transfer, PVDF membranes were kept for blocking using blocking buffer (5% skimmed milk powder in 1x PBST) and incubated over night at 4 o C. After overnight blocking, PVDF membranes were washed with 1x PBST thrice for 3 minutes each. Primary antibodies (polyclonal and monospecific antibodies separately) were diluted (1:100) and PVDF membranes were incubated in diluted primary antibody solution at room temperature with slow shaking on rocker for 2 to 3 hours. PVDF membranes were washed with 1x PBST thrice for 3 minutes each [15, 16] .
Secondary antibody (polyclonal and monospecific antibodies separately) was diluted (1:5000) and PVDF membranes were incubated in diluted secondary antibody solution at room temperature with slow shaking on rocker for 2 to 3 hours. After incubation, PVDF membranes were washed with 1x PBST thrice for 3 minutes each. 12.5 mL Tris buffer (pH 7.35), 30 µl of 30% H2O2, a pinch of DAB were added into detection tray, mixed well and PVDF membranes were kept into the tray. The tray was gently shaken for a period of 10 minutes until the colour developed in the control lane [15, 16] .