263 Wayne F. Anderson (ed.), Structural Genomics and Drug Discovery: Methods and Protocols, Methods in Molecular Biology, vol. 1140, DOI 10.1007/978-1-4939-0354-2_20, © Springer Science+Business Media New York 2014 Chapter 20 Ligand Screening Using Fluorescence Thermal Shift Analysis (FTS) Chi-Hao Luan, Samuel H. Light, Sara F. Dunne, and Wayne F. Anderson Abstract The fluorescence thermal shift (FTS) method is a biophysical technique that can improve productivity in a structural genomics pipeline and provide a fast and easy platform for identifying ligands in protein function or drug discovery screening. The technique has gained widespread popularity in recent years due to its broad-scale applicability, throughput, and functional relevance. FTS is based on the principle that a protein unfolds at a critical temperature that depends upon its intrinsic stability. A probe that will fluoresce when bound to hydrophobic surfaces is used to monitor protein unfolding as temperature is increased. In this manner, conditions or small molecules that affect the thermal stability of a protein can be identified. Herein, principles, protocols, data analysis, and special considerations of FTS screening as performed for the Center for Structural Genomics of Infectious Diseases (CSGID) pipeline are described in detail. The CSGID FTS screen is designed as a high-throughput 384-well assay to be performed on a robotic platform; however, all protocols can be adapted to a 96-well format that can be assembled manually. Data analysis can be performed using a simple curve fitting of the fluorescent signal using a Boltzmann or double Boltzmann equation. A case study of 100 proteins screened against Emerald Biosystem’s ADDit™ library is included as discussion. Key words Protein ligand, Protein folding, Thermal shift, High-throughput screening, Drug discovery 1 Introduction When integrated into a structural genomics pipeline, complimentary biochemical and biophysical techniques can enhance protein struc- ture determination productivity and provide a platform for gener- ating novel insights into protein function. Since the fluorescence thermal shift (FTS) method was first described in 2001, the tech- nique has gained widespread popularity and been effectively applied to address a variety of drug screening and general biophysical ques- tions [1]. Providing an efficient medium- to high-throughput method for monitoring protein thermal denaturation across multiple conditions, FTS has increasingly found a place within structural genomics operations [2–4]. Several features suit FTS for