Case Study: Probing P97 ATPase With the Transcreener ADP² ATPase Assay

Transcreener ATPase Assay Kits provide a sensitive, HTS capable platform as demonstrated in millions of wells and multiple publications. Transcreener is a universal platform eliminating the need for laborious internal assay development.

Kinase Assay ADP FP
Kinase Assay ADP FI
Kinase Assay ADP TR-FRET

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 ATPase Assay Method

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

Universal

The universal nature of the assay 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.

Flexible Assay 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.

Kinetic Mode Capable

Transcreener ATPase Assays 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.

Low Quantity of Enzyme Required

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

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

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

Assay reagents are stable at room temperature for a minimum of 8 hours. The fluorescent signal stability of Transcreener ATPase 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

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