IAP Quantification via Assays

For IAP quantification, IAP and Bradford assays were performed on the cells grown in 24-well plates as previously described (Guo et al., 2017 (link)). The wells were washed with 1× PBS and 200 μl 18 megohm*cm deionized water was added to each well. The plate was sonicated at 4°C for 5 min before transferring the content of each well into different 0.5 ml microcentrifuge tubes. For IAP activity, 85 μl of pNPP solution was added to the required number of wells of a 96-well clear-bottom opaque side plate. To this, 25 μl of prepared samples and IAP standards (prepared using p-nitrophenol and pNPP) were added and allowed to incubate at 37°C for 1 h. For the Bradford assay, 250 μl of Bradford reagent was added to the required number of wells of a 96-well clear-bottom opaque side plate before transferring 5 μl of prepared sample or standards to those wells. The Bradford assay plate was incubated at room temperature for 30 min. The IAP and Bradford assay plates were read at 405 nm and 595 nm, respectively, using a Synergy 2 plate reader, controlled by Biotek's Gen5™ Reader Control and Data Analysis Software.