Hrp conjugated goat anti mouse secondary antibody

The bacterial cultures were diluted to OD₆₀₀ of 0.3 with tris-buffered saline (TBS). The specimens (200 μL) were transferred to a Multiscreen-GV 96-well filter plate, filtered, and washed with TBST buffer (TBS, 0.1% Tween-20). After blocking with with 1% bovine serum albumin (BSA) and 0.01% H₂O₂ in TBST for 1.5 h at 37 °C, and subsequent washing steps, 50 µL of anti-6xHis antibody-horseradish peroxidase (HRP) conjugate (1:200, Thermo Fisher, MA1-80218) was added to each well and incubated for 2 h at 25 °C. For the TFF3 antibody-binding assay, samples were incubated with 50 µL of anti-TFF3 primary antibody (1:450, Sigma, WH0007033M1) for 2 h at 25 °C and washed three times with TBST buffer, followed by incubation of 100 µL goat anti-mouse-HRP conjugated secondary antibody (1:5000, Thermo Fisher, 31430) for 1 h at 25 °C and three subsequent washes. After the wash steps, Ultra-TMB (3,3′,5,5′-tetramethylbenzidine) ELISA substrate (100 µL) was added to each well and incubated for 10 min at 25 °C. To stop the reaction, 50 µL of 2 M sulfuric acid was added to each well. One-hundred microliters of the final reaction was transferred to 96-well plate and measured the absorbance at 450 and 650 nm. The relative amount of displayed peptide was measured by subtracting absorbance at 450 nm with absorbance 650 nm.

Total soluble protein was harvested from HEK293 cells using RIPA buffer with 1× protease inhibitor cocktails (mini-EDTA free) and phosphatase inhibitor cocktails (ThermoFisher Scientific, USA) with western blotting performed as per Conn et al. [26 (link)]. Anti-FLAG M2 antibody (F1804; Sigma-Aldrich USA) was used at 1:2,500 dilution; with goat anti-mouse HRP conjugated secondary antibody (ThermoFisher Scientific, USA) at 1:10,000 dilution. Chemiluminescent detection was carried out using Super Signal West Pico PLUS (ThermoFisher Scientific, USA) and Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standard (Bio-Rad, USA) was used for size estimation.