Protein Kinase Research Reagents
The human kinome consists of 518 protein kinases and 20 lipid kinases, which play crucial roles in a multitude of cellular processes. These processes include cell cycle regulation, metabolism, transcription, cytoskeletal development, cellular motility, apoptosis, cell proliferation, differentiation, and intracellular communication. For more detailed information, you can refer to the publication here.
We offer an extensive portfolio of protein phosphorylation assays, featuring over 300 no-wash assays available in both cell-based and biochemical formats.
Our cellular immunoassays are meticulously designed to provide the high sensitivity required to detect endogenous and basal levels of phosphorylated proteins in cell lysates. This ensures accurate measurement of protein phosphorylation within cells, which is crucial for understanding various cellular processes.
The biochemical assay formats are tailored to facilitate the investigation of kinase activity. These assays enable researchers to screen and identify potent inhibitors for a wide range of kinases, making them invaluable tools for biochemical studies and drug discovery.
In addition, the HTRF kinase binding platform offers a complementary method using three fluorescent inhibitors that cover approximately 80% of the kinome. Instead of detecting compounds that inhibit phosphorylation, this platform detects binding interactions. This approach allows for the detection of inhibitors that bind to kinases with low activity or those that are non-activated.
For research use only. Not for use in diagnostic procedures.
Explore our Protein Kinase solutions
Biochemical kinases
A universal solution: KinEASE assays
Our experts developed specific HTRF™ KinEASE kits to investigate kinase activity, characterize kinases, and screen for inhibitors. The five kits in the product range offer a semi-universal method for addressing phosphorylation on Serine/Threonine and Tyrosine residues, respectively. Over 272 kinases have already been validated with this method.
The principle for these assays is divided into two main steps:
Enzymatic step: During the enzymatic step, the tagged substrate is incubated with the kinase of interest and ATP, allowing the phosphorylation reaction to begin.
Detection step: Detection reagents are added and recognize the phosphorylated substrate. The resulting Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal is proportional to the phosphorylation level.
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- Measuring kinase activity under nearly any condition
- Addressing multiple therapeutic areas with HTRF KinEASE assays
A substrate-specific solution: LANCE™ Ultra kinase assay
LANCE technology (LANthanide Chelate Excitation) Ultra is a solution in which your fluorescently labeled substrate can be chosen specifically. Over 300 kinases were tested and validated with this approach.
The assay protocol is divided in two steps:
- Enzymatic step: The ULight™-peptide substrate is phosphorylated in the presence of kinase and ATP.
- Detection step: The substrate is captured by an Eu-anti-phospho-substrate antibody. Upon excitation, the Eu-chelate transfers its energy to the ULight dye, resulting in fluorescent light emission at 665 nm.
Kinase binding platform
Why choose the HTRF kinase binding assay?
One of the most well-known kinase inhibitors, Gleevec (Imatinib), binds to a non-activated form of Abl kinase. Inhibitors like Gleevec are challenging to detect using activity-based assays. Therefore, setting up kinase binding assays for your kinase of interest is advantageous to ensure you don’t miss out on detecting such compounds.
HTRF kinase binding assays are simple to set up, utilizing a convenient mix-and-measure detection format. These assays do not require the presence of ATP. Instead, they measure the direct displacement of the fluorescent inhibitor from the ATP binding pocket. This allows you to determine the affinity of your inhibitor by performing dose-response curves.
How it works: assay principle
The HTRF Kinase binding assay is based on a biochemical sandwich format. When a tagged kinase is present, an HTRF signal is generated when the tracer is bound to the kinase. Upon adding competitive inhibitors, the fluorescent Staurosporine (or Dasatinib or Sunitinib) is displaced and the HTRF signal disappears.
Kinase inhibitor selection for kinase binding assays
The Revvity discovery kit includes three fluorescent ATP-competitive kinase inhibitors: Staurosporine-red, Dasatinib-red, and Sunitinib-red. We estimate that these three fluorescent inhibitors cover at least 80% of the human kinome.
Our scientists have written three application notes, one per tracer, to guide you in your studies. Each application note provides validation data, guidance for Kd and Ki determination, and case studies.
- HTRF Kinase Binding Assays: Sunitinib-Red Validation
- HTRF Kinase Binding Assays: Dasatinib-Red Validation
- HTRF Kinase Binding Assays: Staurosporine-Red Validation
Kinetic binding parameters: determining Kon and Koff
Dissociation kinetics, and thus inhibitor residence time, have become key parameters for predicting in-vivo drug efficacy outcome. HTRF kinase binding platform assays are ideal for studying the binding kinetics of inhibitors.
Get complete information in this application note entitled, “Kinetic binding of kinase inhibitors and determination of Kon, Koff rates”.
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This HTRF kit allows for the cell-based quantitative detection of Nucleolin when phosphorylated at Thr84.
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Phospho-STAT3 (Tyr705) assay is a sandwich immunoassay for quantitative detection of phospho-STAT3 (Tyr705) in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total STAT1 assay is a sandwich immunoassay for quantitative detection of total STAT1 in cellular lysates using Alpha Technology.
This HTRF kit allows for the cell-based quantitative detection of total B-RAF.
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Phospho-SYK (Tyr525/526) assay is a sandwich immunoassay for quantitative detection of phospho-SYK (Tyr525/526) in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Biotin-Free Human Total SYK assay is a sandwich immunoassay for quantitative detection of total SYK in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total ERK1/2 assay is a sandwich immunoassay for quantitative detection of total ERK1/2 in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Ultra™ Human Phospho-SIRPα (pan-Tyr) assay is a sandwich immunoassay for quantitative detection of phospho-SIRPα (pan-Tyr) in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Phospho-Rb (Thr821) assay is a sandwich immunoassay for quantitative detection of phospho-Rb (Thr821) in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Phospho-JAK2 (Tyr1008) assay is a sandwich immunoassay for quantitative detection of phospho-JAK2 (Tyr1008) in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Ultra™ Human Total AXIN-2 assay is a sandwich immunoassay for quantitative detection of total AXIN-2 in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Ultra™ Human Total PAX5 assay is a sandwich immunoassay for quantitative detection of total PAX5 in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Biotin-Free Human Total Rb assay is a sandwich immunoassay for quantitative detection of total Rb in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total AXIN-1 assay is a sandwich immunoassay for quantitative detection of total AXIN-1 in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total STAT5 assay is a sandwich immunoassay for quantitative detection of total STAT5 in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total PAX8 assay is a sandwich immunoassay for quantitative detection of total PAX8 in cellular lysates using Alpha Technology.
This HTRF kit allows for the cell-based quantitative detection of total Nucleolin.
This HTRF kit allows for the cell-based quantitative detection of LRRK2 when phosphorylated at Ser1292.
This HTRF kit allows for the cell-based quantitative detection of Total LRRK2.
This HTRF kit allows for the cell-based quantitative detection of NPM1 when phosphorylated at Thr199.
This HTRF kit allows for the cell-based quantitative detection of IRAK4 when phosphorylated at Thr345.
This HTRF kit allows for the cell-based quantitative detection of YES when phosphorylated at Tyr426.
This HTRF kit allows for the cell-based quantitative detection of total A-RAF.
The AlphaLISA™ SureFire® Ultra™ Human IRF5 Aggregate assay is a sandwich immunoassay for quantitative detection of IRF5 aggregate in cellular lysates using Alpha Technology.
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) assay is a sandwich immunoassay for quantitative detection of phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) in cellular lysates using Alpha Technology.
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AlphaLISA Di/Mono-Methyl-Histone H3 Lysine 27 (H3K27me2-1) cellular detection kit
AlphaLISA for biomarkers in urine: Measuring the renal tubular injury indicator, β2-microglobulin
