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Transcreener® ADP2 Assays for universal screening and profiling of any ATPase

Ketohexokinase Assay - A Transcreener ADP2 Assay Application

The Transcreener ADP2 Assay is a far-red, competitive assay that measures ketohexokinase (also known as KHK) 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 ketohexokinase modulators. 

This ketohexokinase 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. Ketohexokinase 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 Ketohexokinase Assay Work?

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

Direct Detection of ADP to Measure Ketohexokinase Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener Ketohexokinase 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 Ketohexokinases
  • Screen Compound Libraries for Ketohexokinase 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 ketohexokinase assay is compatible with 96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized KHK 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.

KHK Assay Z' Data

Detection of ADP Under Ketohexokinase 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 500 nM KHK enzyme. Substrate concentrations of 0.15 mM ATP and 7 mM Fructose were 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, 0.01% Triton, 10 mM CaCl2, and 10 mM KCl. The detection mixture included 1X Stop and Detect Buffer, 225 μg/mL ADP2 Antibody, and 8 nM ADP2 Tracer. The KHK enzyme reaction took place for 60 minutes at 30ºC.

KHK Enzyme Titration

Ketohexokinase Assay Enzyme Titration

Linear Response

Linear Response Data with Ketohexokinase

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 22 nM KHK enzyme, 0.15 mM ATP, 7 mM Fructose, 25 mM Tris (pH 7.5), 10 mM MgCl2, 10 mM CaCl2, 10 mM KCl, 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 (Ketohexokinase Inhibitor)

Dose Response Curve with KHK Inhibitor

IC50 = 0.29 μM

Ketohexokinase 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 KHK 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
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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
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SpectraMax® M2/M2eNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidatedClick Me ArrowValidated
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Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableClick Me ArrowValidatedNot Capable
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What's Included

What You Will Need

Assay Plates for TR-FRET

The Role of Ketohexokinase As a Therapeutic Target

Ketohexokinase phosphorylates in the fructose metabolic pathway. The fructose metabolic pathway is involved in metabolic disorders. Studies show KHK loss-of-function mutations lead to accumulating fructose in plasma and urinary excretion, and genetic deletions decrease harmful metabolic effects from fructose that lead to obesity, insulin resistance, hyperlipidemia, and more (Gutierrez et al, 2021). 

As such, ketohexokinase inhibitors show potential to inhibit the fructose metabolic pathway, making it a target of interest for metabolic syndrome. Accelerate your discovery of KHK inhibitors with the Transcreener Ketohexokinase Assay.

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