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Enzyme thermostability analysis

EB Elizabeth L. Bell
GR Gloria Rosetto
MI Morgan A. Ingraham
KR Kelsey J. Ramirez
CL Clarissa Lincoln
RC Ryan W. Clarke
JG Japheth E. Gado
JL Jacob L. Lilly
KK Katarzyna H. Kucharzyk
EE Erika Erickson
GB Gregg T. Beckham
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The thermostabilities of the NylC-TS variants were determined using differential scanning fluorimetry (DSF). For each protein, a 5 µM sample of protein was prepared in reaction buffer and SYPRO Orange dye (provided at a 5000X concentration from the manufacturer), added to a final concentration of 10X. DSF was carried out using a Bio-Rad CFX Connect 96-Real Time PCR system, using the FRET channel for excitation and emission settings, and CFX Manager (version 2.0) for data collection. The temperature was increased with an increment of 0.3 °C/s from 25 °C to 95 °C. The Tm was then determined from the peak of the first derivative of the melt curve from three replicate measurements.

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