Learn More About Transcreener Method Used for this MAPK8 Assay.

Transcreener ADP2 MAPK8 Assay

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.

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 Transreener 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

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

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 Data

Linear Response

MAPK8 Linear Response Curve Data

MAPK8 Enzyme Titration (P38 Substrate)

MAPK8 Enzyme Titration With P38 Substrate Data

MAPK8 Enzyme Titration (ATF2 Substrate)

MAPK8 Enzyme Titration With ATF2 Substrate Data

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 Dose Response Curve Data

Three Fluorescent Readout Options

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

Fluorescent Intensity

MAPK8 Assay FI Readout Data

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 Data

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

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
VANTAstar validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

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.

TOP