Icycler5 pcr instrument

Differential scanning fluorimetry (DSF) is a high throughput method that monitors the thermal unfolding of proteins in the presence of a fluorescent dye (Niesen, Berglund & Vedadi, 2007 (link)). Prior to DSF, purified EC and LT GAPDH were spun through a G50 Sephadex column, pre-equilibrated in DSF buffer (100 mM potassium phosphate, pH 7.0, 150 mM NaCl), at 2500 rpm for 1 min. Protein samples were then aliquoted to a concentration of 0.05 µg/µL/well into the wells of a 48-well, thin-walled PCR plate along with the dye SYPRO Orange (5X final concentration of the dye). PCR plates were then sealed with sealing tape and placed into a BioRad iCycler5 PCR instrument. SYPRO Orange (Invitrogen) fluorescence was monitored as described by Biggar and colleagues (2012 (link)). Briefly, SYPRO Orange was excited through the transmission of light through the FAM filter (485 ± 30 nm), with the subsequent emission of light through the ROX filter (625 ± 30 nm). Measurements were taken every 30 s in 1 °C increments from 25 °C to 97 °C. Subsequent analysis of the fluorescent data using OriginPro 8.5 and the Boltzmann distribution curve yielded the midpoint temperature of the protein-unfolding transition, known as the protein melting temperature (T_m), for GAPDH.

In a 96-well, thin-walled white PCR plate, 5 μl of LiAS-A (2.4 μM) were mixed with 5 μl of 10x SYPRO Orange (λ_exc 485 nm; λ_em 625 nm) and 40 μl of water or the ligands and ligands combinations to be tested. Plates were then sealed and placed into a BioRad iCycler5 PCR instrument. Measurements were taken every minute in 0.5°C increments from 25° to 95°C. Subsequent analysis of the fluorescent data using Biorad iCycler iQ Optical System Software Version 3.1 yielded the protein melting temperature (T_m) for LiAS-A.