An effective thiol-reactive probe for differential scanning fluorimetry with a standard real-time polymerase chain reaction device

Abstract

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the Kd values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT-PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL L-cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT-PCR machines in a hydrophilic or hydrophobic environment.

Keywords: BODIPY FL l-cystine; Differential scanning fluorimetry; High-throughput assay; Membrane proteins; Standard RT–PCR device; Thiol-reactive fluorescent probe.