Transcreener® ADP² Kinase Assays

This application note by BellBrook Labs describes how the Transcreener ADP² Kinase Assay allowed rapid assay development to enable screening and dose-response measurements for five kinases involved in innate immunity.

This application note describes the validation of the Tristar2 S LB 942 Multidetection microplate reader with the Transcreener® Fluorescence intensity assay from BellBrook Labs. The TriStar2 S combines the user friendliness of a multimodal optical system, with the sensitivity and performance of a dedicated optical device. It has been developed for full modularity and equipped with the proprietary ONE-4-ALL optical system.

This application note describes the validation of the Mithras2 LB 943 Monochromator Multimode reader with the Transcreener® Fluorescence intensity assay from BellBrook Labs. The Mithras² LB 943 is a high-end microplate multimode reader based on monochromator technology with excellent performance. Characterized by its sensitivity and robustness, especially in luminescence and BRET measurements, the reader supports all important reading technologies.

This poster from BellBrook Labs looks at alternative fluorescence modes when measuring ADP formation for screening drugs. These methods can be used with most plate readers used today. It also includes a new EZ protocol showing a linear relationship between ATP Concentration and ADP2 Antibody concentration.

In this study the utility of ADP detection as a means to screen inhibitor potency using a far-red, time resolved Förster resonance energy transfer (TR-FRET) format is described. This assay uses the widely adopted TR-FRET technique to take advantage of low compound interference common to this method and owed to its time-gated nature as signal is measured after most background fluorescence has subsided. This assay has proven both sensitive and robust with Z´>0.7 at low substrate conversion levels enabling enzymology during initial velocity conditions and is an important addition to the HTS toolkit.

This poster describes a systematic approach to determine the residence time of compounds using Abl1 and Aurora C as targets and several well characterized ATP-competitive drugs. Residence time can be determined by equilibrium binding measurements using methods such as surface plasmon resonance (SPR), which provides highly quantitative data but requires costly instrumentation and long experimental timelines. Transcreener® ADP2 Assays offer a highly sensitive and versatile method for estimating drug residence times for kinases using a multimode fluorescent plate reader.

ADP detection is an attractive approach for screening kinases and other ATP-utilizing enzymes because it provides a universal platform that can be used for any member of the kinase super family as well as many other ATP-dependent enzymes, regardless of the acceptor substrate. The three ADP detection approaches that have been developed into commercial HTS assay products are 1) direct immunodetection of ADP, which relies on antibodies that selectively recognize ADP in the presence of excess ATP, 2) enzyme-coupled detection, where the ADP is used to drive a cascade of detection enzymes that ultimately produces a fluorescent signal and 3) enzyme-coupled detection, where the residual ATP is first depleted and then ADP is converted to ATP and detected using luciferase. This poster compares the three methods with respect to assay principle, protocol and performance.

The Transcreener ADP2 Assay is a single addition, homogenous assay method that directly measures ADP formation in kinase or ATPase reactions. The first generation Transcreener ADP assay, introduced in 2006 as a far red fluorescence polarization (FP) assay, was recently improved by incorporating a new antibody (ADP2) that enables a 20-fold increase in sensitivity. Together these Transcreener ADP assays have been validated for high throughput screening with diverse targets including protein, lipid and carbohydrate kinases, carboxylases, and chaperonin ATPases. This poster compares these three assays with other nucleotide detection assay methods

Transcreener® HTS is a universal, high throughput biochemical assay platform based on the detection of nucleotides, which are formed by thousands of cellular enzymes - many of which catalyze the covalent regulatory reactions that are central to cell signaling and are of great value as targets in drug discovery. The Transcreener® ADP2 FP Assay is a far-red, competitive fluorescence polarization (FP) assay based on the detection of ADP and is a simple, one-step homogenous detection assay. This Application Note describes how to streamline the process of inhibitor profiling with the Transcreener® ADP2 FP Assay eliminating the need to experimentally optimize the ADP2 antibody concentration and the need for a standard curve.

Transcreener® is a universal, high throughput biochemical assay based on detection of nucleotides, which are formed by thousands of cellular enzymes — many of which catalyze the covalent regulatory reactions that are central to cell signaling and are high value targets in drug discovery. This application note describes the use of the Transcreener® assay in the determination of Km for ATP and Kemptide with Protein Kinase A Enzyme.

Watch this webinar from Meera Kumar, Senior Scientist at BellBrook Labs, to learn more about CD73 as a target for immunotherapy, including information on how the transcreener CD73 assay was developed and how this assay can be used to help measure adenosine.

The grant will be used to accelerate the discovery of new treatments for autoimmune diseases by targeting the cGAS-STING pathway

With this addition, researchers can more easily screen for small molecule modulators of cGAS

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