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Learn More About Transcreener Method Used for this MAPK8 Assay.

MAPK8 Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Assay directly measures ADP produced by Mitogen-Activated Protein Kinase 8 (known as MAPK8 or JNK1). These ADP measurements allow researchers to effectively determine the activity of MAPK8 enzyme. The assay provides a powerful tool to screen compound libraries for MAPK8 modulators to help find new therapies for disease.

The kit comes complete with the detection reagents required to measure activity. MAPK8 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

Application Note - Detection of MAPK8 Activity with the Transcreener ADP2 Kinase Assay

This application note describes methods for using the Transcreener ADP2 Kinase Assay to measure MAPK8 enzymatic activity in a high throughput (384-well) format, e.g., for inhibitor screening and/or inhibitor dose-response measurements. 

Application Note Preview Image

How Does This MAPK8 Assay Work?

The Transcreener MAPK8 Assay determines MAPK8 enzyme activity by directly measuring the ADP formed by the enzyme using the Transcreener ADP Assay. By detecting ADP output, the assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time. 

Transcreener MAPK8 Assay technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.

The MAPK8 assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.

Direct Detection of ADP to Measure MAPK8 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener MAPK8 Assay Schematic

The workhorse. Used in many large screens. Best deck and signal stability.

Fluorescent Intensity (FI)

ADP FI Kinase Assay Kit

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

ADP TR-FRET Kinase Assay Kit

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure Enzymatic Activity of MAPK8
  • Screen Compound Libraries for MAPK8 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled ADP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP, FI, or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The MAPK8 assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized MAPK8 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. 

MAPK8 Robust Assay Data

MAPK8 Under Initial Velocity Conditions - FP Readouts

The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 5 µM ATP. Linearity is shown here using C-Jun as a substrate under initial velocity conditions when raw data is converted to ADP formed. The enzyme buffer includes 50 mM Tris pH 7.5, 10 mM MgCl2, 0.05% BSA, and 0.01% Brij. The Detection mix included 20 μg/mL ADP2 antibody, 4 nM ADP Alexa 633 tracer, and 1X Stop & Detect Buffer B. The MAPK8 enzyme reaction took place for 60 minutes at room temperature. The Detection Mix was added and incubated for 40 minutes at room temperature and read with a CLARIOstar plate reader.

MAPK8 Enzyme Titration (C-Jun Substrate)

MAPK8 Enzyme Titration With C-Jun Substrate

Linear Response

MAPK8 Assay Linear Response Curve C-Jun Substrate

MAPK8 Enzyme Titration (P38 Substrate)

MAPK8 Enzyme Titration With P38 Substrate

MAPK8 Enzyme Titration (ATF2 Substrate)

MAPK8 Enzyme Titration With ATF2 Substrate

Screen for Inhibitors & Perform SAR

Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow-up SAR can also be performed using the assay to determine inhibitor potency with ease. Assay conditions include 0.150 μg/mL MAPK8, 50 mM Tris, 0.01% Brij-38, 10 mM MgCl2, 0.05% BSA, and 5 μM ATP. The Detection Mix consists of 1X Stop & Detect Buffer B, 20 μg/mL ADP Ab, and 4 nM 633 tracer. The compound was incubated with enzyme for 30 minutes prior to starting the enzymatic reaction at room temperature. 

Dose-Response Curve

MAPK8 Assay Dose Response Curve

IC50 = 0.25 μM

Three Fluorescent Readout Options

Choose the readout that is the best fit for your lab based on preference and plate reader compatibillity.

Fluorescent Intensity

FI Detection Mix (10 μL): 1x Stop & Detect Buffer B, 400 μg/mL ADP Ab-IRDye QC1, 8 nM AlexaFluor 594 Tracer. 

TR-FRET

MAPK8 Assay TR-FRET Readout

TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer C, 8 nM ADP Ab-Tb, 2000 nM ADP HiLyte647 tracer.

MAPK8 Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your MAPK8 program.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Far-Red FP, FI & TR-FRET Readouts Validated on Major Multimode Readers

SupplierInstrumentFP AssaysFI AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidatedClick Me ArrowValidated
Tristar 5In ReviewClick Me ArrowValidatedIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidatedClick Me ArrowValidated
CytationTM 5ValidatedValidatedValidated
CytationTM 3ValidatedValidatedValidated
CytationTM 1ValidatedValidatedValidated
SynergyTM H1ValidatedValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableValidatedNot Capable
SynergyTM Neo 2ValidatedValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewClick Me ArrowValidatedIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M2/M2eNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact UsClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedIn ReviewClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableClick Me ArrowValidatedNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowValidatedClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated

What's Included

What You Will Need

The Role of MAPK8 As a Therapeutic Target

MAPK8 is a key mediator in cellular responses induced by extracellular signals. MAPK8s are involved in a spectrum of cellular processes, including cell proliferation, differentiation, and apoptosis. Loss-of-function studies show MAPK8 has essential roles in modulating immune cell function, development of the nervous system, and functions in the CNS (Yarza et al, 2016).

Since MAPK8s play a fundamental role in regulating key biological processes, it is not surprising that aberrant MAPK8 is associated with cancer. MAPK8 activation was often reported in multiple cancer cell lines and patient tissue samples (Kennedy et al, 2003). However, studies have also found that MAPK8 acts as a tumor suppressor for certain forms of cancer and tumor microenvironments. Because of these correlations, MAPK8 has become an attractive therapeutic target for cancer treatments (Bubici et al, 2014). 

Transcreener ADP² MAPK8 Assay is an excellent tool for researchers examining therapeutic effects of MAPK8 inhibitors.

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