Case Study: Probing P97 with the Transcreener ADP² ATPase Assay

Discover ATPase Inhibitors with the Transcreener ADP2 Assay

Transcreener ATPase Assay Kits provide a sensitive, HTS capable platform as demonstrated in millions of wells and multiple publications. As a universal technology eliminating the need for laborious internal assay development, Transcreener directly measures ADP produced by enzymatic ATP hydrolysis. Direct detection provides a simple yet robust method, eliminating coupling enzymes while providing accurate results.

Ultra-Sensitive Assay Measures ATPase Activity

In this peer-reviewed article, learn how researchers at Cancer Research UK used the sensitivity of Transcreener as an HSP90 and HSP72 activity assay when the malachite green technique wasn't sensitive enough for their drug discovery.

ATPase Assay Principle - Direct ADP Detection with a Fluorescent Readout

ATPases are a group of enzymes often found in cell membranes that catalyzes the decomposition of ATP. As the enzyme hydrolyzes ATP, it yields a free phosphate ion, ADP, and energy. Sensitive detection of ADP by the Transcreener antibody displaces a tracer producing a fluorescent readout.

Three detection modes are available - you can choose between FP, FI or TR-FRET readouts.  All three assays use far-red fluors, ensuring that interference from fluorescent library compounds and light scattering is reduced to insignificant levels.

There are a number of reasons for choosing one over the other: minimizing compound interference, maximizing instrument performance, comparison to previous data, or just familiarity and comfort level. Choose the one the best fits your research requirements.

FP ADP ATPase Assay
FI ADP ATPase Assay
TR-FRET ADP ATPase Assay

Applications

  • Screen compound libraries for enzyme inhibitors
  • Determine inhibitor selectivity between enzymes
  • Profile hits for inhibitor potency
  • Measure enzymatic activity
  • Determine residence times for inhibitors

Fewer False Positives

Direct detection of ADP reduces the degree of interference seen with coupling enzyme-based assays, lowering the number of false positives. Far-red tracers eliminate fluorescent compound interference. Reduce your time triaging false hits and spend it finding druggable leads.

Non Radio-Active Assay Method

Transcreener provides an accurate, dependable, safe alternative to radio-labeled assays. No more disposal concerns.

Universal

The universal nature of Transcreener allows for the screening of virtually any ATPase enzyme. Universal assays also provide an effective technique for difficult targets. Get ahead of the curve with assays ready for emerging targets.

High Throughput Assay in a Flexible Format

Use with 96 well, 384 well, or 1536 well plates depending on your desired format.

Mix-and-Read Simplicity

Fewer steps mean faster results, limited interference, less robot programming, and a lower probability for error.

Biochemical ATPase Assay Steps

Kinetic Mode Capable

Transcreener ADP² Kits can be used in kinetic mode, allowing the assay to be used in applications not possible with endpoint assays methods. With this capability, you can measure the residence times of inhibitors with a simple biochemical assay to pick leads with the greatest potential to become drugs. See how you can measure drug-target residence times in this free 12-page article, A Guide to Measuring Drug-Target Residence Times with Biochemical Assays.

Low Quantity of Enzyme Required

The high affinity of the ADP antibody generates sensitive and robust detection of ADP down to 10 nM with Z’ values of 0.7 or greater. As a result, less enzyme is required for your experiments.

p97 ATPase Enzyme Titration

The ADP FP Assay window at 10% conversion (0.5 µM ADP). The recommended enzyme concentration was an EC80 of 20 ng/mL. The assay demonstrated a Z' of 0.87 and a lower limit 0f detection at 0.06 μM ADP under these conditions.

Sensitive Initial Velocity Detection Over a Broad ATP Range

Easily detects less than 10% ATP conversion using 0.1 to 1,000 µM ATP, assuring accurate inhibitor potencies and minimizing enzyme and substrate costs.

ADP ATPase Assay Detection Modes v6

ADP/ATP standard curves for a broad range of initial ATP concentrations (0.1 μM to 1000 μM ATP) using the FP, TR-FRET and FI assay respectively. ADP was increased as total ATP + ADP was held constant. Antibody was adjusted based on a linear relationship between EC85 and initial ATP. All assays were performed in 384-well plates (n=24) and read on the Tecan Safire 2 (FP), BMG Labtech PHERAstar Plus (TR-FRET), or the Perkin Elmer EnVision (FI) multimode plate readers.

Stable Reagents and Assay Signal

Reagents are stable at room temperature for a minimum of 8 hours. The fluorescent signal stability of Transcreener ADP² Assays lasts at least 24 hours. This provides the flexibility necessary to perform large automated screens.

Far Red FP, FI & TR-FRET Readouts Validated on Major Multimode Readers

Transcreener ADP² ATPase Assay Kits

*For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

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