Infinite 200 rx plate reader

Sample wells were coated with 10 μg of TMV and the control wells were coated with only 1 X TBS. The plate was incubated at room temperature for 1 h with shaking at 400 rpm. The plate was later blocked with 5% (w/v) BSA for 1 h with shacking at 400 rpm. At the meantime, 0.01 μg of TBP_T25-biotin was incubated with a serial percentage of BSA (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) for 1 h with shaking at 450 rpm. The plate was washed once with 0.1% TBST for 5 min followed by adding in the pre-blocked TBP_T25-biotin. The plate was incubated for 1 h with shaking at 400 rpm. After washing thrice with 0.1% TBST (5 min each time), streptavidin HRP conjugate (Abcam ab7403, diluted 1:10,000 in 5% (w/v) BSA) was added and incubated at room temperature for 1 h, shaking at 400 rpm. After further washes in 0.1% TBST (3 × 5 min) we added 100 μL of the tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) to each well. The plate was incubated in the dark for ~10 min and the absorbance was measured at 450 nm using an Infinite 200 Rx plate reader (Tecan Life Sciences) with 25 flashes in 96-well flat-bottom plate mode. Equation [inhibitor] vs [response] from GraphPad Prism was used to calculate the IC₅₀ of the BSA inhibition. Paired t-test was used to compute the significance difference of the two observations.

We coated each well of a Nunc Maxisorp flat-bottom 96-well plate with 10 μg TMV (in 0.1 M bicarbonate buffer, pH 8.6) and incubated overnight at 4 °C. Plates coated with 5% (w/v) BSA was used as negative controls. Next day, plates were blocked with 5% (w/v) BSA and incubated at room temperature for 1 h, shaking at 800 rpm. The plates were then washed with 0.1% TBST (3 × 1 min). We added 20 μL of amplified phage from each biopanning cycle to each well in 5% (w/v) BSA and incubated at room temperature for 1 h, shaking at 800 rpm. Plates were then washed with 0.5% TBST (3 × 5 min) followed by the addition of 100 μL HRP-conjugated anti-M13 monoclonal antibody (Abcam ab50370, diluted 1:500) and incubation at room temperature for 1 h, shaking at 800 rpm. After further washes in 0.5% TBST (3 × 5 min) we added 100 μL of the tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) to each well. The plates were incubated in the dark for 10 min and the absorbance was measured at 370 nm using an Infinite 200 Rx plate reader (Tecan Life Sciences) with 25 flashes in 96-well flat-bottom plate mode.