Contents

2.1 Introduction 25

2.2 Substrate Finding Approaches 27

2.3 Assay Formats 28

2.3.1 Fluorescence Intensity 28

2.3.2 Fluorescence Resonance Energy Transfer (FRET) 31

2.3.2.1 FRET Quench 32

2.3.2.2 Time-Resolved (TR) FRET 35

2.3.3 Fluorescence Polarization (FP) 36

2.3.4 Fluorescence Lifetime 39

2.4 Kinetic Parameters 41

2.4.1 Determination of Kinetic Parameters KM and kcat under Initial Velocity Conditions 42

2.5 Flow Chart for Assay Development 42

2.6 Summary and Conclusion 43

References 45

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