title: 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.
authors: 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.
authors: Lukas
authors: L
authors: Hofmann
authors: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors: Sahil
authors: S
authors: Gulati
authors: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors: Avery
authors: A
authors: Sears
authors: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors: Phoebe L
authors: PL
authors: Stewart
authors: 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.
authors: Krzysztof
authors: K
authors: Palczewski
mesh_terms: D001896:Boron Compounds / Q000737:chemistry*; D003553:Cystine / Q000031:analogs & derivatives* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry; D057927:Hydrophobic and Hydrophilic Interactions; D011506:Proteins / Q000032:analysis* / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013696:Temperature
chemical_list: C000612044:BODIPY-FL L-cystine; D001896:Boron Compounds; D005456:Fluorescent Dyes; D011506:Proteins; D013438:Sulfhydryl Compounds; D003553:Cystine
keywords: BODIPY FL l-cystine; Differential scanning fluorimetry; High-throughput assay; Membrane proteins; Standard RT–PCR device; Thiol-reactive fluorescent probe