Disadvantages of Metabolic Reduction Assays

Some of the major disadvantages of both the tetrazolium and resazurin reduction assays are related to the requirement to incubate a substrate with viable cells for a sufficient time to generate a measurable signal. This is a very important feature to consider when designing assays for HTS. In addition to the extra plate handling steps needed to return the cells to a 37 C incubator, the extended incubation of the detection reagents with viable cells increases the possibility of undesirable...

Introduction

Proteases are enzymes catalyzing the hydrolysis of peptide bonds. They form one of the largest enzyme families encoded by the human genome, with more than 500 active members. Based on the different catalytic mechanisms of substrate hydrolysis, these enzymes are divided into four major classes serine threonine, cysteine, metallo, and aspartic proteases. In serine, cysteine, and threo-nine proteases, the nucleophile of the catalytic site is a side chain of an amino acid in the protease (covalent...

Fluorescence Lifetime

Fluorescence lifetime (FLT) is a recent addition to the portfolio of readouts for biochemical protease assays. Lifetime is an intrinsic property that corresponds to the average time fluorophore electrons remain in the excited state before relaxing to the ground state. The FLT assay principle is described in Figure 2.7. In the case of a single emitting species, the probability of observing a photon at a certain time point after the fluorophore is excited follows an exponential decay (Moger et...

Determination of Kinetic Parameters Km and kcat under Initial Velocity Conditions

The Michaelis constant, KM, can be obtained from measurements conducted at a constant enzyme concentration and different substrate concentrations. Under conditions in which the substrate concentration S is significantly higher than the enzyme concentration and the substrate consumption is below 20 , the initial enzymatic velocity v0 can be approximated by the Henri-Michaelis-Menten equation where vmax is the maximal velocity at saturating substrate concentrations. The initial enzyme velocity is...

Fluorescence Polarization FP

FP is an alternative readout principle for endopeptidase activity assays. FP or anisotropy measurements allow the detection of changes in the rotational correlation time of particles. These differences in the rotational correlation (or relaxation) time are related to different masses of particles. The experimental determination of steady-state fluorescence anisotropy requires the linear polarization of the light used for the excitation of the probe as well as linear polarization of the emitted...

Radioactive Assay Technologies

The earliest assays were based on the use of 32P as a label either on the ATP cofactor for kinases or on a peptide substrate for phosphatases. With kinases, the transfer of the 32P from the g position of ATP to a peptide or protein substrate resulted in a 32P-labeled peptide or protein that would be separated away from the ATP by capture on a filter and subsequent washing. The quantity of phos-phoprotein could be quantified by scintillation counting. The availability of a new version of...

Lb

4> & & A< 3 p W V cf FIGURE 4.5 Development of automated electrophysiology assay for KV1.3. (A) Description of IonWorks process for KV1.3 assay (PP PatchPlate). (B) Distribution of pre-DMSO and post-DMSO K+ current amplitudes in typical PatchPlate PPC. (C) Distribution of pre- and post-compound seal resistances in typical PatchPlate PPC. (D) Family of K+ currents recorded from a representative single well of a PatchPlate PPC. Currents were evoked by a series of 300-ms test pulses,...

Flow Chart For Assay Development

The starting point of all successful assay development processes is a protein preparation that ideally contains only the enzyme of interest. The amount of enzyme needed for the whole assay development process depends mainly on the catalytic efficiency of the enzyme with the substrate. Only 10 of a protease can be sufficient for proper determination of the kcatIKM value, the determination of the endpoint linearity by an enzyme titration, and the assay validation by IC50 determination for a...

Contents

About the Chapter 1 Assay Development for Protein Kinases and Chapter 2 Fluorescence-Based Biochemical Protease Assay Chapter 3 Assay Development for Nuclear Hormone Chapter 4 Assay Development for G Protein-Coupled Receptors and Ion Channels 59 Joseph G. McGivern, Yi-xin Qian, and Paul H. Lee Chapter 5 Assay Development for Heat Shock Chapter 6 Assay Development for Cell Viability and Apoptosis for High-Throughput Terry L. Riss, Richard A. Moravec, and Andrew L. Niles Chapter 7 Assay...

References

Substrate specificity of recombinant osteoclast-specific cathepsin K from rabbits. Biol. Pharm. Bull. 19, 1026-1031. Andrews, D.L. and Demidov, A.A. 1999. Resonance Energy Transfer. Chichester John Wiley amp Sons. Angliker, H. et al. 1995. Internally quenched fluorogenic substrate for furin. Anal. Biochem. 224, 409-412. Attucci, S. et al. 2002. Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates. Biochem. J....

Development of Scintillation Proximity Assay for GPR23

GPR23 P2Y9 is a novel receptor that binds lysophosphatidic acid LPA , although its physiological importance is not fully understood Noguchi, Ishii, and Shimizu 2003 . In order to develop a GPR23 binding assay, we prepared membranes from a Chinese hamster ovary CHO cell line that expressed human GPR23 under the control of a tetracycline-inducible expression system. Under normal conditions, these cells expressed GPR23 at a very low level but could be induced by tetracy-cline or a derivative to...

Cellular Enzyme Linked Immunoadsorbent Assay ELISA

The technique of cellular ELISA is to treat the cells with a test compound, and by modulating kinase or phosphatase activity, cause a change in a particular signaling pathway. The change in phosphorylation state of the signaling components can be detected by lysing the cells and measuring the phosphorylation state of the analytes by capturing them between an anti-analyte antibody and an anti-phosphopeptide or anti-phosphotyrosine antibody Minor, 2003 . The ELISA plates are coated with the...