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Case Study: Probing p97 ATPase with the Transcreener ADP2 Assay

p97 Assay - A Transcreener ADP2 Assay Application

The Transcreener ADP2 Assay is a far-red, competitive assay that measures p97 activity via direct detection of ADP produced during an enzyme reaction. It is designed for high-throughput screening, with a single-addition, mix-and read format. The assay is an excellent tool for researchers screening compound libraries to discover p97 modulators. 

This p97 assay provides excellent data quality (Z' ≥ 0.7) and signal (≥ 85 mP polarization shift) at low substrate conversion (typically 10% or less). It accommodates ATP concentrations ranging from 0.1 µM to 1,000 µM.

The kit comes complete with the detection reagents required to measure activity. p97 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme. This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

How Does This p97 Assay Work?

The Transcreener p97 Assay determines p97 enzyme activity by using the Transcreener ADP2 monoclonal antibody to directly detect ADP produced during the enzyme reaction. This antibody is highly selective to ADP over ATP. ADP produced in the reaction competes with a far-red fluorescent ADP2 tracer, changing the fluorescent properties and providing a fluorescent readout.

The p97 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 p97 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener p97 Assay Schematic

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

Fluorescent Intensity (FI)

ADP FI Kinase Assay Kit

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 Kit

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

Applications

  • Measure Enzymatic Activity of p97
  • Screen Compound Libraries for p97 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
  • Available in FP, FI, or TR-FRET readouts

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

Run your enzyme reaction, add Transcreener reagents, and read your plates. The p97 assay is compatible with 96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized p97 reaction conditions indicate a robust assay.  Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z' shown here = 0.94.

p97 Z' Data

Detection of ADP Under p97 Initial Velocity

Assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we performed a 12 point, 2-fold dilution starting at 250 nM p97 enzyme. A concentration of 0.15 mM ATP was used. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The buffer included 25 mM Tris pH 7.5, 10 mM MgCl2, and 0.01% Triton. The detection mixture included 1X Stop and Detect Buffer, 225 μg/mL ADP2 Antibody, and 8 nM ADP2 Tracer. The p97 enzyme reaction took place for 60 minutes at 30ºC.

p97 Enzyme Titration

p97 Assay Enzyme Titration

Linear Response

Linear Response Data p97 Titration

Screen for Inhibitors & Perform SAR

Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow-up SAR can also be performed using the assay to determine inhibitor potency with ease. Assay conditions include 8.5 nM p97 enzyme, 0.15 mM ATP, 25 mM Tris (pH 7.5), 10 mM MgCl2, and 0.01% Triton for 60 minutes at 30ºC. The Detection Mix included 225 μg/mL ADP2 Antibody, 8 nM ADP2 Tracer, and 1X Stop and Detect Buffer.

Dose-Response (NMS-873)

Dose Response Data with p97 Assay

IC50 = 17.08 nM

p97 Assay Services

BellBrook Labs offers ATPase Profiling Services for several ATPase enzyme targets using our trusted Transcreener ATPase Assay. BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your ATPase program. We tailor our expertise to meet your specific needs, saving you both time and money. Accelerate your p97 program with our ATPase profiling services.

Scientist Studying DDX41

ATPase Profiling 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.
  • Triaging Non-stoichiometric Inhibitors - Run assays under different conditions such as varying detergent and enzyme concentrations.

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

SupplierInstrumentFP AssaysFI AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidatedClick Me ArrowValidated
Tristar 5In ReviewClick Me ArrowValidatedIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidatedClick Me ArrowValidated
CytationTM 5ValidatedValidatedValidated
CytationTM 3ValidatedValidatedValidated
CytationTM 1ValidatedValidatedValidated
SynergyTM H1ValidatedValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableValidatedNot Capable
SynergyTM Neo 2ValidatedValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewClick Me ArrowValidatedIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M2/M2eNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact UsClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedIn ReviewClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableClick Me ArrowValidatedNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowValidatedClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated

What's Included

What You Will Need

Assay Plates for TR-FRET

The Role of p97 As a Therapeutic Target

p97 has been associated with a variety of diseases, including neurodegenerative diseases, cancers, and viral infections. It plays an important role as a regulator of endoplasmic reticulum-associated degradation (ERAD), mitochondrial-associated degradation, chromatin-associated degradation, and more. Over-expression of p97 correlates to many cancers, while p97 mutations correlate to many neurodegenerative disorders (Huryn et al, 2020). 

Because of its correlation to several viruses, cancers, and neurodegenerative disorders, researchers search for p97 modulators as targets for these diseases. The Transcreener p97 Assay makes screening and profiling for modulators easy and accurate in order to accelerate the discovery of therapeutic treatments.

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