Protein Kinase Research Reagents
The human kinome comprises 518 protein kinases and 20 lipid kinases. These proteins are involved in a wide variety of cellular processes, including cycle mechanisms, metabolism, transcription, cytoskeletal development, cellular motility, apoptosis, cell proliferation, differentiation, and even intracellular communication, see the publication here.
We offer an extensive protein phosphorylation portfolio of no-wash assays available in cell-based or biochemical formats. Our assays provide the sensitivity required to detect endogenous and basal levels of Phosphorylated proteins in cell lysates or allow kinase activity investigation to select potent inhibitors for over 300 kinases in biochemical assays.
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 ULightTM dye, resulting in fluorescent light emission at 665 nm.
Cell based phospho/total detection
Intracellular signaling pathways, also called signal transduction cascades, play a key role in sensing and integrating diverse external stimuli to generate biological responses. The stimuli are detected by a cell surface receptor which transmits the initial signal inside the cell. There, it is then multiplied by a transduction cascade mediated by the phosphorylation of multiple protein substrates.
Protein phosphorylation is a dynamic process, tightly controlled by kinase and phosphatase cascades, which regulates many cellular processes and has been identified as an underlying mechanism for a variety of diseases.
The Cell Signaling kits developed by Revvity are specifically intended for the direct measurement of proteins (phosphorylated and/or total) within cells.
HTRF assay principle
HTRF cellular protein phosphorylation kits offer a rapid and streamlined alternative to long-standing technologies such as Western blot, ELISA, and other luminescent bead-based assays
Following treatment, cells receive a lysis treatment leading to the release of intracellular proteins, both phosphorylated or unphosphorylated, from the cell’s compartment. Released proteins are detected by addition of the reagents.
All HTRF phospho and total assays are based on a sandwich immunoassay format:
- Phospho specific and total antibodies, labelled with Cryptate or d2, are used for the quantification of phosphorylated proteins
- Two different antibodies, labelled with Cryptate or d2, are used for the quantification of total proteins.
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AlphaLISA™ SureFire® Ultra™ assay principle, Multiplex SureFire Ultra and Terbium SureFire Ultra
AlphaLISA SureFire Ultra assay kits allow the rapid, sensitive, and quantitative detection of phosphoproteins from cells. The AlphaLISA SureFire Ultra assay kits are optimized for enhanced signal-to-noise windows.
AlphaLISA assays require two bead types: Donor beads and Acceptor beads. In AlphaLISA SureFire Ultra, the Donor bead is coated with streptavidin to capture the biotinylated antibody while the Acceptor bead is coated with a proprietary "CaptSure™" agent that immobilizes the other antibody which is labeled with a CaptSure tag.
In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor beads in close proximity, whereby the singlet oxygen transfers energy to excite the Acceptor bead, enabling the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
In addition to AlphaLISA SureFire Ultra kits detecting phosphorylated proteins, there is a subset of AlphaLISA SureFire Ultra kits that allow for the detection of the "total" analyte (i.e. non-phosphorylated as well as phosphorylated), offering an ideal way to normalize for positive or negative modulation of protein expression in cellular contexts.
We also offer Multiplex assays, divided into two categories:
- Multiplex SureFire Ultra (MPSU) kits are complete kits for phospho and total detection of the same target. Assessing the total protein level (phosphorylated and non-phosphorylated) is essential in distinguishing whether up- or down-regulation of phosphorylation is due to changes in that protein’s expression levels or to cell viability.
- Terbium SureFire Ultra (TBSU) kits measure a single cell signaling target and are intended to be combined with a standard AlphaLISA SureFire Ultra kit (ALSU) to assess the second target. The TBSU kits allow for a mix-and-match combination of any two targets in the assay lists, providing measurement of two separate targets with flexibility.
Assay workflow
A cell signaling experiment has 4 steps illustrated below:
Read more details for each technology in the respective guides:
Kinase binding platform
You must have heard about the best-known kinase inhibitor, Gleevec. This inhibitor binds a non-activated form of Abl and is therefore more difficult to detect by activity-based assays. We’re now introducing our Kinase binding platform.
Dont miss potentially promising compounds anymore!
How to proceed
Revvity’s different assays enable you to perform compound screening, pharmacological studies, or determine the targeted inhibitor’s IC50 value or inhibition constant (Ki). The assay workflow is based on three stages.
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The ultimate guide to successful kinase binding experiments:
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, 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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