PTP1B enzymatic assay

The PTP1B inhibitory assay was carried out using a slight modification to literature reported method¹⁸. p-nitrophenyl phosphate (pNPP) was used as a substrate for the measurement of enzyme activity. Buffer solution consisted of 25 mM Tris–HCl (pH 7.5), 2 mM β-mercaptoethanol, 1 mM (ethylenediaminetetraacetic acid) EDTA, and 1 mM dithiothreitol (DTT). The assay was performed by adding 10 μL test compound solution to 20 μL of enzyme (1 μg/ml), and then mixing with 40 μL of 4 mM pNPP in 130 μL of the given buffer using 96 well plate at 37 °C for 10 min. During the enzymatic reaction, pNP was produced as a result of pNPP dephosphorylation which was monitored by Spectra Max M3 multi-mode microplate reader at 405 nm for 30 min. NaVO₄ was used as positive control for inhibition. The experiments were carried out in triplicate, and the samples concentration needed to inhibit 50% of enzyme activity under the assay conditions were defined as the IC₅₀ values. Lineweaver–Burk and Dixon plots methods were used to determine kinetic parameters. Sigma Plot (SPCC Inc., Chicago, IL) was used to calculate these parameters.