Open AccessAnalysis of Protein Stability and Ligand Interactions by Thermal Shift Assay
Author(s) -
Huynh Kathy,
Partch Carrie L.
Publication year - 2015
Publication title -
current protocols in protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.409
H-Index - 32
eISSN - 1934-3663
pISSN - 1934-3655
DOI - 10.1002/0471140864.ps2809s79
Subject(s) - recombinant dna , thermal stability , chemistry , ligand (biochemistry) , denaturation (fissile materials) , protein stability , fluorescence , target protein , high throughput screening , chromatography , biophysics , biochemistry , biology , organic chemistry , receptor , physics , quantum mechanics , nuclear chemistry , gene
Purification of recombinant proteins for biochemical assays and structural studies is time‐consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real‐time PCR instruments. By screening a wide range of solution conditions and additives in a 96‐well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low‐cost screen to discover new protein‐ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small‐scale, high‐throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. © 2015 by John Wiley & Sons, Inc.