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.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The ketohexokinase assay is compatible with 96, 384, and 1536-well formats.
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.
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.
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.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration (in the Detection Mixture) for FP is 225 μg/mL for 500 μM ATP. Yours may vary depending on, enzyme, and buffer composition. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| ATP and ADP | ATP and ADP are used to create the ATP/ADP standard curve. ATP is used as a phosphate donor to start the reaction. We suggest using a starting ATP concentration of 0.15 mM for ketohexokinase. |
| Component | Notes |
|---|---|
| Ketohexokinase Enzyme | We have successfully used recombinant human Ketohexokinase protein from R&D Systems Cat. #8177-HK. A ketohexokinase concentration of 22 nM was used for screening and SAR. |
| Substrate | We have successfully used D-(-)-Fructose from Sigma-Aldrich Cat. #F0127. |
| Enzyme Buffer | 25 mM Tris (pH 7.5), 10 mM MgCl2,, 10 mM CaCl2, 10 mM KCl, and 0.01% Triton. Enzyme buffer components may be optimized as required. |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 FI - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the ADP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
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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