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Amplite® Colorimetric Enterokinase Activity Assay Kit
Enterokinase dose response was measured with Amplite® Colorimetric Enterokinase Activity Assay Kit on a 96-well clear bottom microplate using a SpectraMax microplate reader (Molecular Devices) with path check on mode.
Protein kinases (or simply kinases) are intracellular enzymes known as phosphotransferases that regulate cell growth, proliferation, and immunity. Kinases have the ability to phosphorylate a number of proteins, whose method is colloquially termed O-phosphorylation, and action can trigger a number of intracellular signaling cascades, especially in pathways related to signal transduction. Kinases also assume a crucial role in other mechanistic cellular processes including protein synthesis, cell division, cell development, apoptosis, and aging.

Roughly 500 kinase genes have been identified, representing close to 2% of the human genome, and kinase activity has been found to alter up to 30% of the human proteome. Biochemically, the phosphate (P) from kinases are transferred from an adenosine triphosphate (ATP) group to specific residues on proteins. The reaction between kinases and, namely, serine, threonine, or tyrosine accounts for over a third of all protein phosphorylation events. To a lesser extent, histidine and aspartate metabolites may also be N-phosphorylated. As adenosine diphosphate (ADP) is a byproduct of kinase activity, ATP and ADP are both common targets in kinase activity assays and arrays.

Kinases may be programmed or deactivated in a variety of ways, including autophosphorylation, or by binding with an activator or inhibitor protein. Kinase dysregulation is not simply limited to cancer, but is also seen in inflammatory conditions and neurodegeneration. Some specific diseases associated with kinase dysregulation and overexpression include diabetes mellitus, hypertension, Parkinson's and Alzheimer's disease.


Types of Human Protein Kinases

  • AGC kinases: containing PKA, PKC and PKG.
  • CaM kinases: containing the calcium/calmodulin-dependent protein kinases.
  • CK1: containing the casein kinase 1 group.
  • CMGC: containing CDK, MAPK, GSK3 and CLK kinases.
  • STE: containing the homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases.
  • TK: containing the tyrosine kinases.
  • TKL: containing the tyrosine-kinase like group of kinases.


Kinases & P53 Receptor

Kinase phosphorylation has been specifically noted to stimulate the p53 receptor, a sequence-specific transcription factor that regulates an abundant number of downstream target genes that may cease gene replication, halt the cell cycle, start DNA repair, or even induce apoptosis. Experiments with the p53 receptor may also include using one or more of the known inhibitors for some procedures.

Chronic inactivation of the p53 receptor protein due to an excess in phosphorylation or dephosphorylation events has been proven to potentially turn a cell cancerous.

P53 regulation and signaling

Simplified pathways and related effects of P53 regulation and signaling, including phosphorylation steps. Figure created in BioRender.


Common Methodologies

Radiometric Kinase Activity Assay

Traditionally, radiometric means have been used to assess kinase activity. These protocols utilize a 32P-labeled ATP, and steps may be modified for use with immunoprecipitated cultured cells or with recombinant affinity-tagged purification over a wide range of reaction conditions.
  1. A kinase reaction buffer is made using a protein inhibitor, a reducing agent, and ATP
  2. The reaction buffer and kinase sample are combined and incubated
  3. A stop solution is added
  4. SDS-PAGE is performed
The gel apparatus must be shielded to limit the exposure of 32P, and is stained, dried, and produced into an autoradio diagram. The autoradiograph can provide a qualitative visualization of results, and data is expressed in terms of relative radioactivity.

Note: A Geiger counter is commonly used to check the intensity of the radiographic signal, though other tools may also be used to identify the levels of ionizing radiation, including dosimeters or scintillators.

Upon analysis, the specific activity of the kinase of interest (typically, in units of nanomole phosphate transferred per min per mg kinase) may be calculated. Additionally, the number of moles of phosphate incorporated in the substrate may also be determined to identify the number of phosphosites on a particular protein.

Fluorescent Detection

A number of fluorescent methods exist that can be used to detect kinase activity that avoid the use of radioactivity. Many commercial kits are available that use simple fluorescence endpoint measurement techniques where fluorescent probes attach to and label peptide substrates.

Most kits exploit the fact that ADP is produced from a kinase phosphorylation, and use this to directly determine enzyme activity. ADP is then detected and quantified by evaluating fluorescent intensity of the reaction sample. These assays can be evaluated in real time, and offer a powerful tool to screen compound libraries for kinase modulators which may be useful to identify new therapies for disease.

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Principle of a standard fluorescent polarization assay
Principle of a standard fluorescent polarization assay. Figure made in BioRender.
Another technique is known as fluorescent polarization (FP). In FP, an inhibitor is first added to the kinase sample. Next, a tracer and antibody are added to the reaction mix and the antibody associates with either the labeled tracer or the kinase-produced phosphorylated substrate. Kinase activity can be analytically graphed as a function of concentration versus polarization, where the amount of antibody bound to the tracer is inversely proportional to the amount of the phosphorylated substrate present.

Table 1. Common FRET donor and acceptor pairs and their R0 values.

IAEDANS*5-IAF (5-Iodoacetamidofluorescein)49

In time-resolved (TR) fluorescence resonance energy transfer (FRET), a donor fluorophore is conjugated to an antibody and a phosphorylated product of a specific kinase reaction is labeled with an acceptor fluorophore. The labeled antibody specifically binds to the labeled phosphorylated product, and this interaction brings the donor and acceptor fluorophores into close proximity where resonance energy transfer can occur. FRET analysis can be performed by measuring changes in the intensity of the sensitized demission of the acceptor probe and/or by measuring the decrease in the fluorescence of the donor probe over its lifetime.
A fourth method exists called fluorescence lifetime microscopy (FLIM) that is an imaging technique that utilizes the differences in the exponential rate of decay of the photon emission of a fluorophore from a sample. The general principle of FLIM is to measure the average time that the fluorescent molecules spend in an excited state, known as the fluorescent lifetime. FLIM analysis is a versatile technique that can map the spatial distribution of lifetimes with microscopy in fixed or living cells. FLIM can also be used to indirectly measure molecular concentrations and interactions that are closely related to the fluorescence lifetime of the fluorophores.

Luminescent Detection

PhosphoWorks™ Luminescence ATP Assay Kit *DTT-Free *
Jurkat cell number was measured with the PhosphoWorks™ Luminescence ATP Assay Kit *DTT-Free * on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The kit can detect as low as 100 cells. The integration time was 1 sec.
Assays that use ADP luminescence can alternatively provide a highly sensitive, high-throughput screening method to measure kinase activity.

Most luminescent-based assays proceed in simple steps:
  1. The kinase reaction is allowed to proceed
  2. A stop reagent is added to halt the kinase reaction and deplete any remaining ATP
  3. A detection reagent is added that converts ADP to ATP
  4. After incubation, newly synthesized ATP can be measured using a luciferase-luciferin reaction
Methodology follows other similar luminescent assay protocols, and analysis can be performed via a plate-reader luminometer and/or charge-coupled device (CCD) camera. Luminescence can be correlated to ADP concentrations by using an ATP-to-ADP conversion curve, created in parallel with the assay that functions as the experimental standard curve.

These assays are not necessarily specific to kinases, and can be used to monitor the activity of other ADP-generating enzymes, like ATPase.

Qualitative Polymerase Chain Reaction (qPCR)

three-step amplification procedure
Like conventional PCR, qPCR utilizes the same three-step amplification procedure - denaturation, annealing, and extension (figure made in BioRender).
Qualitative polymerase chain reaction (qPCR) is an ultrasensitive technique that can quantitate multiple kinases on a single panel with high throughput capabilities. qPCR works by detecting the associated DNA label, and comparing it to known controls to measure the amount of kinase captured in test samples.

Table 2. Double-stranded DNA-binding dyes for qPCR

Ex (nm)
Em (nm)
Unit Size
Cat No.
Helixyte™ Green *20X Aqueous PCR Solution*498 nm522 nm5x1 mL17591
Helixyte™ Green *10,000X Aqueous PCR Solution*498 nm522 nm1 mL17592
Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*490 nm525 nm1 mL17597
Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*490 nm525 nm10 mL17598
Q4ever™ Green *1250X DMSO Solution*503 nm527 nm100 µL17608
Q4ever™ Green *1250X DMSO Solution*503 nm527 nm2 mL17609

qPCR methods are multifaceted and have a dynamic range of ability. They may also be used to determine dissociation constants for kinase interactions that can be calculated by measuring the amount of kinase captured as a function of kinase concentration within a test sample. It has also been reported for detecting type I, type II, and allosteric kinase inhibitors. qPCR does not experimentally require ATP, so only true thermodynamic interaction affinities are measured, as opposed to other techniques that may rely on IC50 values.

Sandwich ELISA

Sandwich ELISA offers the quantitative determination of kinases activity using either adherent, suspension cell culture extracts, or purified enzyme preparations. Most kits utilize a kinase substrate coated onto a 96-well plate, a phosphotyrosine linked horseradish peroxidase (HRP) antibody, and an HRP substrate (TMBZ), but a wide variety of options are available.

These techniques provide a rapid, sensitive method to monitor and assess kinase activity, and for detecting substrate specificity in samples. Sandwich ELISAs are also nonradioactive, cost-effective, and can be completed in 1.5-2 h using simple colorimetric detection.

Table 3. Available HRP Substrates

Abs/Ex (nm)
Ex (nm)
Unit Size
Cat No.
ReadiUse™ ABTSChromogenic420 1 L11001
ReadiUse™ TMBChromogenic650 100 mL11012
ReadiUse™ TMBChromogenic650 1 L11003
Amplite® ADHPFluorogenic57158425 mg11000
Amplite® BlueFluorogenic32440925 mg11005
Amplite® RedFluorogenic5715841000 Assays11011
Amplite® IRFluorogenic6486681 mg11009
LuminolChemiluminescent3554121 g11050

Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

LC-MS/MS are phosphoproteomic methods that involve the highly selective enrichment of phosphopeptides. LC-MS/MS enables the identification of protein phosphorylation without bias, and the observability of kinase activity depends on the amount of the phosphorylated proteins and phosphosites present in a sample.

In general, the first step in the procedure involves:
  1. Mixing the proteins extracted from cells, or their digested peptides, with a recombinant protein kinase
  2. Next, the reaction mixture is digested with a specific proteinase or peptidase
  3. After enrichment, the reaction sample produces a phosphopeptide that may be measured by LC-MS/MS
LC-MS/MS offers large-scale identification of phosphorylated peptides and phosphosite localization, which can provide information about the significance between kinase activity and physiology.


Product Ordering Information


Table 4. Related Kinase Products for Protein Kinase Activity

Product Name
Unit Size
11410Amplite® Colorimetric Enterokinase Activity Assay Kit200 Tests
31001Amplite® Universal Fluorimetric Kinase Assay Kit *Red Fluorescence*250 Tests
31005ReadiUse™ Universal Kinase Reaction Stopping Buffer100 mL
8A0459Aurora Kinase (Phospho-Thr288) Antibody-
8A0531p70 S6 Kinase (Phospho-Thr229) Antibody-
8A0532p70 S6 Kinase (Phospho-Ser371) Antibody-
8A0533p70 S6 Kinase (Phospho-Thr389) Antibody-
8A0534p70 S6 Kinase (Phospho-Ser418) Antibody-
8A0761Fructose 6 Phosphate Kinase (Phospho-Ser483) Antibody-
8A0783Breast Tumor Kinase (Phospho-Tyr447) Antibody-
8A1192Casein Kinase II α (Phospho-Tyr255) Antibody-
8A1194Casein Kinase I α (Phospho-Tyr321) Antibody-
8A1224PI3-kinase p85-α/γ (Phospho-Tyr467/199) Antibody-
8A1225PI3-kinase p85-α (Phospho-Tyr607) Antibody-
8A7073p44/42 MAP Kinase (Phospho-Thr202) Antibody-
8A7074p44/42 MAP Kinase (Phospho-Tyr204) Antibody-
8A7188p70 S6 Kinase (Phospho-Ser411) Antibody-
8A7189p70 S6 Kinase (Phospho-Ser424) Antibody-
8A7190p70 S6 Kinase (Phospho-Thr421) Antibody-
8A8157p70 S6 Kinase β (Phospho-Ser423) Antibody-
8B0459Aurora Kinase (Ab-288) Antibody-
8B0531p70 S6 Kinase (Ab-229) Antibody-
8B0532p70 S6 Kinase (Ab-371) Antibody-
8B0533p70 S6 Kinase (Ab-389) Antibody-
8B0534p70 S6 Kinase (Ab-418) Antibody-
8B0761Fructose 6 Phosphate Kinase (Ab-483) Antibody-
8B0783Breast Tumor Kinase (Ab-447) Antibody-
8B1192Casein Kinase II α (Ab-255) Antibody-
8B1224PI3-kinase p85-α/γ (Ab-467/199) Antibody-
8B1225PI3-kinase p85-α (Ab-607) Antibody-
8B7073p44/42 MAP Kinase (Ab-202) Antibody-
8B7074p44/42 MAP Kinase (Ab-204) Antibody-
8B7188p70 S6 Kinase (Ab-411) Antibody-
8B7189p70 S6 Kinase (Ab-424) Antibody-
8B7190p70 S6 Kinase (Ab-421) Antibody-
8B8156p70 S6 Kinase (Ab-427) Antibody-
8B8157p70 S6 Kinase β (Ab-423) Antibody-
8C101516-Phosphofructo-2-Kinase Antibody-
8C10708Hexokinase-3 Antibody-
8C10868Mevalonate Kinase Antibody-
8C11007Breast Tumor Kinase Antibody-
8C11030Glucokinase Regulator Antibody-



Importance of Protein Kinase and Its Inhibitor: A Review
The active kinome: The modern view of how active protein kinase networks fit in biological research
Fluorescence detection techniques for protein kinase assay
Assaying Protein Kinase Activity with Radiolabeled ATP
Rapid, ELISA-based measurement of protein tyrosine kinase activity using the K-LISA™ Kit
Recent advances in methods to assess the activity of the kinome
Cytochrome C Oxidase