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

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

  • Anal Biochem. 2016 Apr 15;499:63-65. doi: 10.1016/j.ab.2016.01.016.
Lukas Hofmann 1,  Sahil Gulati 2,  Avery Sears 2,  Phoebe L Stewart 2,  Krzysztof Palczewski 3
Affiliations

Affiliations

  • 1 Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address: lxh278@case.edu.
  • 2 Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
  • 3 Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address: kxp65@case.edu.
PMID: 26851339 DOI: 10.1016/j.ab.2016.01.016
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.

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