From growth to proliferation and differentiation on to metabolism and eventually death, kinases control nearly every process in cellular physiology. They initiate major signaling cascades by simply transferring phosphates to specific serine, threonine, or tyrosine residues of their substrates. These consequential events change the substrate conformation, which modifies its binding capacity, catalysis, and recruitment of downstream effectors that make up cellular signaling pathways. These pathways activate gene transcription, switch on or off specific cell-surface receptors, or regulate protein-protein interactions that dictate how a cell lives and dies. Abnormalities in phosphorylation can contribute to many different disease states. It is for this reason that keen interest has been placed in developing compounds to control kinase activity. Also needed are assays that can detect kinase activity and their subsequent modulation by inhibitory compounds. Cayman offers more than 1,200 kinase inhibitors for use in research to understand basic kinase mechanics and disease states. To help with the discovery of novel kinase inhibitors, we offer assays to measure the functional activity of specific kinases. Kinase antibodies as well as their phosphorylated counterparts and proteins are available to help study kinases in your experimental system. Here we provide an overview of the methodologies available from Cayman to assess kinase activity. 

Cell-Based Reporter Assays

Cayman partners with INDIGO Biosciences to deliver their line of cell-based receptor assays. The kinase reporter assays include frozen, ready-to-use human reporter cells that are transfected with vectors containing key regulatory elements of downstream intracellular signaling proteins associated with a specific kinase. A luciferase gene is functionally linked to these regulatory elements. Its activation will reveal the result of an upstream stimulus or inhibition that alters transcription of the reporter. The principal application of this type of assay is to screen compounds to quantify any functional activity, either inhibitor or activator, that they may exert against the kinase. Quantifying changes in luciferase activity in compound-treated reporter cells versus untreated reporter cells provides a sensitive surrogate measure of compound-induced changes in upstream kinase activity.

Reporter cell assay workflow. Reporter cells are dispensed into the wells of the assay plate and incubated for 4-6 hours. Following the preincubation period, culture media is discarded, and the treatment media is added. Following 22-24 hours of incubation, treatment media is discarded, and the luciferase detection reagent is added. The intensity of light emission (in units of relative light units; RLU) from each assay well is quantified using a plate-reading luminometer.

The reporter cells are carefully cryopreserved to remain healthy and division competent. Upon thawing, they can be immediately dispensed into assay plates without rinsing, spinning, determining viability, or adjusting titers before the assay is set up. Since the cells are already transfected, the user does not need to have molecular biology skills to create a vector, allowing the user to immediately resuspend the cells and introduce the treatment. The assay kits include two different optimized media—one for recovering the cryopreserved cells and one for diluting test samples—along with a positive control, luciferase detection reagents, and a cell culture-ready assay plate.

Kinase Reporter Assays

Human Transforming Growth Factor Beta Receptors I/II Reporter Assay System
Human Epidermal Growth Factor Receptor 1 Reporter Assay System
Human Vascular Endothelial Growth Factor Receptor 2 Reporter Assay System

TR-FRET Assays

Our TR-FRET assays measure intracellular signaling events in cell lysates by detecting the target of kinase activity: phosphorylated and/or total levels of specific substrates of the kinase. The platform uses a traditional sandwich immunoassay principle in a homogenous assay format that does not require washing or separation steps. Once cells are treated with experimental compounds and lysed, the protein of interest is detected with a pair of fluorophore-labeled antibodies—one labeled with a europium chelate donor fluorophore and one labeled with a far-red acceptor fluorophore—that both detect distinct epitopes on the readout protein. Some kits offer companion antibody pairs to detect both phosphorylated and total target protein levels. When the europium-labeled antibody and the far-red-labeled antibody are both bound to this protein, a high TR-FRET signal is observed. Potential inhibitors are discovered based on their ability to reduce target phosphorylation and subsequent phospho-specific antibody binding, resulting in a decreased TR-FRET signal. Depending on the assay selected, this method can be used to study active kinases by assessing the role of phosphorylation.

TR-FRET assay workflow. Cells are seeded in a culture plate overnight then treated with stimulator or inhibitor for a pre-optimized period. Cell culture medium is removed, and cells are lysed in buffer. Cell lysates are transferred to a TR-FRET detection plate and either phospho-protein antibody mix or total-protein antibody mix is added to the appropriate assay wells. The assay is read on a TR-FRET-compatible plate reader at two wavelengths to detect both the emission from the europium chelate donor fluorophore at 615 nm and the acceptor fluorophore at 665 nm.

This assay platform is compatible with both adherent and suspended cells. The simple add-incubate-measure protocol requires only single-step reagent addition, which streamlines the time needed to complete the assay. TR-FRET assays have very low background fluorescence levels and high signal-to-background ratios. Ratiometric reporting of the signal output increases the assay reproducibility and robustness.

TR-FRET Assays

pan-Akt (Phospho-Ser⁴⁷³) TR-FRET Assay Kit
pan-Akt (Total) and pan-Akt (Phospho-Ser⁴⁷³) TR-FRET Assay Kit
pan-Akt (Phospho-Thr³⁰⁸) TR-FRET Assay Kit
pan-Akt (Total) TR-FRET Assay Kit
pan-Akt (Total) and pan-Akt (Phospho-Thr³⁰⁸) TR-FRET Assay Kit
EGFR (Phospho-Tyr¹⁰⁶⁸) TR-FRET Assay Kit
EGFR (Total) TR-FRET Assay Kit
EGFR (Total) and EGFR (Phospho-Tyr¹⁰⁶⁸) TR-FRET Assay Kit
ERK1/2 (Phospho) TR-FRET Assay Kit
ERK1/2 (Total) TR-FRET Assay Kit
ERK1/2 (Total) and ERK1/2 (Phospho) TR-FRET Assay Kit
Insulin Receptor β (Phospho-Tyr^1150/1151) TR-FRET Assay Kit
SLP-76 (Phospho-Ser376) TR-FRET Assay Kit
SLP-76 (Total) TR-FRET Assay Kit
SLP-76 (Total) and SLP-76 (Phospho-Ser376) TR-FRET Assay Kit
STAT3 (Phospho-Tyr⁷⁰⁵) TR-FRET Assay Kit
STAT3 (Total) TR-FRET Assay Kit
STAT3 (Total) and STAT3 (Phospho-Tyr⁷⁰⁵) TR-FRET Assay Kit
STAT5 (Phospho-Tyr^694/699) TR-FRET Assay Kit
STAT5 (Total) TR-FRET Assay Kit
STAT5 (Total) and STAT5 (Phospho-Tyr^694/699) TR-FRET Assay Kit
eIF2α (Phospho-Ser⁵¹) TR-FRET Assay Kit
eIF2α (Total) TR-FRET Assay Kit
eIF2α (Total) and eIF2α (Phospho-Ser⁵¹) TR-FRET Assay Kit

Kinase Inhibitor Screening or Activity Assays

Our sphingosine kinase (SPHK) inhibitor screening assays measure the effects of compounds on this specific kinase activity by directly quantifying its phosphorylated substrate, sphingosine. These assays use a kinase-specific fluorogenic substrate (NBD-sphingosine). When SPHK phosphorylates this substrate in the presence of ATP, the shift in spectral properties can be quantified using a fluorescence plate reader.

SPHK1 inhibitor screening assay workflow. SPHK1 and NBD-sphingosine are mixed in the assay buffer and added to the wells. NBD-sphingosine appears golden yellow in color. Serial concentrations of inhibitor or control compounds are added to the appropriate wells, then ATP is introduced. After a 60-minute incubation, the plate is read with an excitation wavelength of 550 nm and an emission wavelength of 584 nm.

The addition of an effective inhibitor will suppress this spectral shift. This assay principle can be used to rapidly screen large inhibitor libraries and is suitable for the characterization of novel inhibitors. These kits conveniently package the kinase, substrate, ATP, and buffer and include a potent kinase inhibitor as a positive control to ensure proper performance.

Inhibitor Screening/Activity Assay

Sphingosine Kinase 1 Inhibitor Screening Assay Kit
Sphingosine Kinase 2 Inhibitor Screening Assay Kit

Phospho-Specific Antibodies and Recombinant Kinases

The development of phosphorylation state-specific antibodies has revolutionized some traditional and more cumbersome methods to directly measure protein phosphorylation. These antibodies can be used in immunocytochemistry/immunohistochemistry, Western blot, or to develop ELISAs to identify the activated kinase of interest. Because the measured levels of a phosphorylated substrate may change with treatment or be altered through experimental errors, a companion antibody that detects the total level of the kinase target regardless of phosphorylation state is useful for determining phosphorylated levels relative to total levels. Recombinant proteins are also useful for Western blot, ELISA, or enzyme assay applications. Each of Cayman's recombinant kinases are engineered in the optimal expression system and characterized for purity, stability, and functionality. Many are available with affinity tags to facilitate detection or further purification.

Phospho-specific antibodies detect only the phosphorylated forms of proteins in a complex protein mixture within cells. They recognize the phosphorylated amino acids in the context of the surrounding amino acid sequence.

Antibodies

Proteins

These functional approaches offer valuable tools for kinase drug discovery to help identify novel inhibitors. They can be used to understand the biological impact of kinase signaling and also serve as tools for drug discovery optimization. Cayman offers more than 1,200 kinase inhibitors along with a focused Kinase Screening Library and Comprehensive Kinase Screening Library to complement these assay technologies. We also offer custom services, including the development of novel assays and the synthesis of novel compounds, to support your specific research interests. Contact us to learn more.

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