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.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.
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.
Performing the MAPK1 titration within the initial velocity region (<20% conversion of substrate to product) confirms that the signal is within dynamic range for 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.
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.
Three fluorescent readout options are available. Choose a readout detection mode best suited for your lab based on preference and plate reader compatibility.
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 Detection Mix (10 μL): 1x Stop & Detect Buffer C, 8 nM ADP Ab-Tb, 17.3 nM ADP HiLyte647 tracer.
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.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration for 5 μM ATP (in the Detection Mixture) for FP - 6.4 μg/mL, TR-FRET - 8 nM, and FI - 5.4 μg/mL . Yours may vary depending on enzyme and buffer composition. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. The suggested concentration for 5 μM ATP (in the Detection Mixture) for FP - 4 nM, TR-FRET - 17.3 nM, and FI - 8 nM. |
| Stop & Detect Buffer B | The Stop & Detect Buffer components will stop enzyme reactions that require Mg2+. To ensure that the enzyme reaction is stopped completely, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction. |
| ATP and ADP | ATP and ADP are used to create the ATP/ADP standard curve. ATP is used as a phosphate donor to start the reaction. We suggest using a starting ATP concentration of 5 μM for MAPK1. |
| Component | Notes |
|---|---|
| MAPK1 Enzyme | We have successfully used recombinant human MAPK1 protein from ThermoFisher Cat. #PV3595. A MAPK1 concentration of 2 nM was used for screening and SAR. |
| Enzyme Buffer | 20 mM Tris (pH 7.5), 10 mM MgCl2,, 1 mM DTT and 0.01% Tween-20. Enzyme buffer components may be optimized as required. |
| APRTPGGRR Peptide | MBP Peptide Substrate [APRTPGGRR] from Anaspec Cat. #AS-27168. |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 FI - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the ADP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
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BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
