Sypro orange protein thermal shift dye

A cell-permeable ATP-competitive kinase inhibitor library from Selleckchem (Houston, TX, United States; catalog No. L1200) was screened to identify interactors for ZmDRIK1-KD. One micromolar of each puri ed construct of ZmDRIK1-KD was mixed with DSF buffer (100 mM K-phosphate, 150 mM NaCl, 10% glycerol, and pH 7.5) containing 1:1000 SYPRO Orange Protein Thermal Shift Dye (Life Technologies Corporation, Eugene, USA) in a 384-well plate containing 10 µM of each library compound. As compound stocks were stored at 10 mM in 100% DMSO, a control with 0.1% DMSO was used as a reference. Plates were sealed using optically clear lms, and uorescence intensity data were measured in a temperature gradient from 25 to 95°C at a constant rate of 0.05°C/s in a QuantStudio 6 qPCR instrument (Applied Biosystems, Singapore). Data were analyzed using the Boltzmann function. Compounds with increased melting temperature by 2° C or more, in comparison to the control curve, were considered positives.

A cell-permeable ATP-competitive kinase inhibitor library from Selleckchem (Houston, TX, United States; catalog No. L1200) was screened to identify interactors for ZmDRIK1-KD. One microgram of each purified construct of ZmDRIK1-KD was mixed with DSF buffer (100 mM K-phosphate, 150 mM NaCl, 10% glycerol, and pH 7.5) containing 1:1000 SYPRO Orange Protein Thermal Shift Dye (Life Technologies Corporation, Eugene, USA) in a 384-well plate containing 10 µM of each library compound. As compound stocks were stored at 10 mM in 100% DMSO, a control with 0.1% DMSO was used as a reference. Plates were sealed using optically clear films, and fluorescence intensity data were measured in a temperature gradient from 25 to 95°C at a constant rate of 0.05°C/s in a QuantStudio 6 qPCR instrument (Applied Biosystems, Singapore). Protein melting temperature was calculated based on the Boltzmann function fitting to experimental data, as implemented in the Protein Thermal Shift Software (Applied Biosystems, Singapore). Compounds displaying a positive temperature shift (ΔTm) higher than 2°C compared to the control were considered positive.