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Application Note: Detection of MAPK1 Activity with the Transcreener ADP2 Assay

MAPK1 Assay - A Transcreener ADP2 Assay Application

The MAPK1 assay leverages the Transcreener ADP2 Assay, a competitive immunoassay, to enable researchers to assess enzymatic activity. The assay relies on direct ADP detection, where binding of tracer to antibody causes change in fluorescence. It is designed for inhibitor screening and inhibitor dose response measurements. 

MAPK1 (also known as ERK2) mediates a variety of cellular responses through phosphorylation of diverse substrates; it is part of the RAF-MEK-ERK pathway which plays a critical role in regulating cell growth, differentiation, and survival and is upregulated in many types of cancers. The search for small molecule MAPK1 modulators is a promising strategy for immunoncology therapeutics. 

The kit includes all necessary detection reagents for measuring activity. Note that MAPK1 enzyme is not provided in the assay kit. For questions regarding enzyme acquisition, please contact us. This assay is designed for use with purified enzyme preparations and is not validated for application with cell lysates, blood, serum, or other biological samples.

Directly Detect ADP to Measure MAPK1 Activity with Three Fluorescent Readout Options

Fluorescent Polarization (FP)

Transcreener MAPK1 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 MAPK1
  • Screen Compound Libraries for MAPK1 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Residence Time Measurements

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

A Simple Mix-&-Read Format

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

Transcreener Mix and Read Assay

Robust Data for Sizeable HTS (Z' = 0.90)

Robust data like this confirms the MAPK1 assay is well-suited for screening large compound libraries. A Z' > 0.7 indicates an assay with clear distinction between active and inactive compounds in a screen. 

Z' Data for MAPK1 Assay

Validation of MAPK1 Assay Under Initial Velocity Conditions

Performing the MAPK1 titration within the initial velocity region (<20% conversion of substrate to product) confirms that the signal is within the dynamic range for the optimized Transcreener ADP2 Assay. The assay show linearity when raw data is converted to ADP using a standard curve. Here, the buffer included 20 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT, and 0.01% Tween-20. The MAPK1 enzyme reaction took place for 60 minutes at room temperature. The detection mixture included 1X Stop and Detect Buffer B, 6.4 μg/mL ADP2 Antibody, and 4 nM ADP2 Tracer. The detection mix was added and incubated for 60 minutes at room temperature.

MAPK1 Enzyme Titration

MAPK1 Assay Titration in FP Readout

Linear Response

MAPK1 Linear Conversion Data Complete Only

Interrogate Novel MAPK1 Modulators in a High Throughput Format

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 here included 2 nM MAPK1 enzyme, 20 mM Tris (pH 7.5), 10 mM MgCl2, 1 mM DTT, 5 μM ATP, 0.01% Tween-20, 6.4 μg/mL ADP2 Antibody, 4 nM ADP2 Tracer, and 1X Stop and Detect Buffer.

Dose-Response Data With Staurosporine

Dose Response Data with MAPK1 Assay

IC50 = 0.77 μM

Choose a Fluorescent Readout Option Best Suited For Your Lab

Three fluorescent readout options are available. Choose a readout detection mode best suited for your lab based on preference and plate reader compatibility.

Fluorescent Intensity

MAPK1 Titration in FI Readout

FI Detection Mix (10 μL): 1x Stop & Detect Buffer B, 5.4 μg/mL ADP Ab-IRDye QC-1, 8 nM AlexaFluor 594 Tracer.

TR-FRET

MAPK1 Enzyme Titration in TR-FRET Readout

TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer C, 8 nM ADP Ab-Tb, 17.3 nM ADP HiLyte647 tracer.

MAPK1 Assay Services

BellBrook Labs offers Lead Discovery Services for MAPK1 using our trusted Transcreener ADP2 Assay. BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your program. We are a small, custom shop so we can tailor our expertise to meet your specific needs, saving you both time and money. 

BellBrook Scientist Performing Lead Discovery 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.
  • Triaging Non-stoichiometric Inhibitors - Run assays under different conditions such as varying detergent and enzyme concentrations.
  • Evaluate Compound-Target Binding - Run thermal shfit assays to confirm compound-target engagement via shifts in melting temperature.

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
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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
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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
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