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

Lukas Hofmann; Sahil Gulati; Avery Sears; Phoebe L. Stewart; Krzysztof Palczewski

doi:10.1016/j.ab.2016.01.016

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

Lukas Hofmann, Sahil Gulati, Avery Sears, Phoebe L. Stewart, Krzysztof Palczewski

Case Western Reserve University

Research output: Contribution to journal › Article › peer-review

26 Scopus citations

Abstract

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the K_d 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.

Original language	English
Pages (from-to)	63-65
Number of pages	3
Journal	Analytical Biochemistry
Volume	499
DOIs	https://doi.org/10.1016/j.ab.2016.01.016
State	Published - 15 Apr 2016
Externally published	Yes

Bibliographical note

Publisher Copyright:
© 2016 Elsevier Inc. All rights reserved.

Funding

We thank Yaroslav Tsybovsky, Nathan Alexander, Tivadar Orban, Marcin Golczak, and Leslie T. Webster Jr. for helpful comments on this manuscript. This work was supported by funding from the National Institutes of Health ( EY009339 , K.P.) and the Arnold and Mabel Beckman Foundation . L.H. is supported by the Swiss National Science Foundation Doc.Mobility fellowship ( P1SKP3_158634 ). K.P. is the John Hord Professor of Pharmacology.

Funders	Funder number
National Institutes of Health
National Eye Institute	R01EY009339
Arnold and Mabel Beckman Foundation
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung	P1SKP3_158634

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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10.1016/j.ab.2016.01.016

Cite this

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

Abstract

Bibliographical note

Funding

Keywords

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