Case Study: Probing P97 with the Transcreener ADP² ATPase Assay

Transcreener ADP2 ATPase Assay

Transcreener ADP2 ATPase Assay directly measures ADP produced by ATP hydrolysis without use of coupling enzymes. These ADP measurements allow researchers to effectively determine the activity of the ATPase enzyme. The assay provides a powerful tool to screen compound libraries for ATPase modulators to help find new therapies for disease. 

The kit comes complete with the detection reagents required to measure activity. ATPase enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

Transcreener ATPase Assay kits provides a sensitive, HTS capable platform as demonstrated in millions of wells and multiple publications. In this peer-reviewed article, learn how researchers at Cancer Research UK used the sensitivity of Transcreener ATPase Assay as an HSP90 and HSP72 activity assay when the malachite green technique wasn't sensitive enough for their drug discovery.

How Does This ATPase Assay Work?

The Transcreener ADP Assay determines ATPase enzyme activity by directly measuring the ADP formed by the enzyme during ATP hydrolysis. By detecting ADP output, the assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time.

Transcreener ATPase Assay technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.

The ATPase assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.

Direct Detection of ADP to Measure ATPase Enzymatic Activity

Fluorescent Polarization (FP)

The workhorse. Used in many large screens. Best deck and signal stability.

Fluorescent Intensity (FI)

ADP FI Kinase Assay

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

ADP TR-FRET Kinase Assay

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure enzymatic activity of ATPase
  • Screen compound libraries for ATPase Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled ADP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z'>0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method (no disposal concerns)
  • Available in FP, FI, or TR-FRET readouts

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.

Learn about measuring drug-target residence times in this free article: Guide to Measuring Drug-Target Residence Times with Biochemical Assays.

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.

Universal Method

The universal nature of Transcreener ATPase 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.

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Biochemical ATPase Assay Steps

Run your enzyme reaction, add Transcreener reagents, and read your plates. The ATPase assay is compatible with 96, 384, and 1536-well formats.  

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.

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.

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.

ATPase Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your ATPase program.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

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

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
VANTAstar validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

What's Included

What You Will Need

ATPases As Drug Targets

ATPases are large macromolecular biological catalyst enzymes that catalyze the hydrolysis from ATP to ADP. As the enzyme hydrolyzes ATP, it yields a free phosphate ion, ADP, and energy. Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a principal role in a wide variety of cellular functions.

ATPase inhibitors are attractive molecular targets for drug discovery. A well-known ATPase inhibitor is the popular pharmaceutical, omeprazole. Omeprazole targets the H+/K+-ATPase system by using selective and irreversible inhibition of the proton pump in the gastric parietal cells. They are also targeted for other areas of health and disease because of their wide variety of cellular functions. Finding therapeutic methods to reign in overactivity requires selective technologies that can help assess the ability to interact with the target. Robust, HTS-ready assays that are accurate yet flexible provide a powerful tool to accelerate discovery.

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