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. 2022;2533:247–257. doi: 10.1007/978-1-0716-2501-9_15

Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions.

Sophia Pinz, Eva Doskocil, Wolfgang Seufert
PMCID: PMC9761908  PMID: 35796993

Abstract

Thermofluor is a fluorescence-based thermal shift assay, which measures temperature-induced protein unfolding and thereby yields valuable information about the integrity of a purified recombinant protein. Analysis of ligand binding to a protein is another popular application of this assay. Thermofluor requires neither protein labeling nor highly specialized equipment, and can be performed in a regular real-time PCR instrument. Thus, for a typical molecular biology laboratory, Thermofluor is a convenient method for the routine assessment of protein quality. Here, we provide Thermofluor protocols using the example of Cdc123. This ATP-grasp protein is an essential assembly chaperone of the eukaryotic translation initiation factor eIF2. We also report on a destabilized mutant protein version and on the ATP-mediated thermal stabilization of wild-type Cdc123 illustrating protein integrity assessment and ligand binding analysis as two major applications of the Thermofluor assay.

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