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Learn More About Transcreener Method Used for this RIPK2 Assay.

RIPK2 Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Assay directly measures ADP produced by RIPK2 (Receptor-Interacting Serine/Threonine Protein Kinases) to measure enzymatic activity. RIPK2 inhibitors are promising targets for many autoinflammatory diseases and cancers. This assay provides a powerful tool to screen compound libraries for RIPK2 modulators to help find new disease therapies.

The kit comes complete with the detection reagents required to measure activity. RIPK2 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 RIPK2 Activity with the Transcreener ADP2 Kinase Assay

This application note describes methods for using the Transcreener ADP2 Kinase Assay to measure RIPK2 enzymatic activity in a high throughput format for inhibitor screening and/or inhibitor dose-response measurements.

Application Note Preview Image

How Does This RIPK2 Assay Work?

The Transcreener RIPK2 Assay determines RIPK2 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 RIPK2 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 RIPK2 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 RIPK2 Enzymatic Activity

Fluorescence Polarization (FP)

Transcreener RIPK2 Assay Schematic

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

Fluorescence 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 RIPK2
  • Screen Compound Libraries for RIPK2 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-&-Read, HTS-Ready

Run your enzyme reaction, add Transcreener reagents, and read your plates. The RIPK2 assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized RIPK2 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 = 0.74.

RIPK2 Z' Data

Detection of ADP Under RIPK2 Initial Velocity (FP Readout)

The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 50 μM ATP, 40 mM Tris (pH 7.5), 2.5 mM MnCl2, and 20 mM MgCl2. The enzyme buffer includes 40 mM Tris, 0.05 mM DTT, and 0.1 mg/mL BSA. The Detection Mix included 1X Stop & Detect Buffer B, 40 μg/mL ADP Antibody, and 4 nm Alexa 633 tracer. The RIPK2 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.

RIPK2 Enzyme Titration

RIPK2 Assay Enzyme Titration

Linear Response

RIPK2 Linear Response 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 0.12 μM RIPK2, 50 μM ATP, 40 mM Tris (pH 7.5), 2.5 mM MnCl2, 20 mM MgCl2, 0.05 mM DTT, and 0.1 mg/mL BSA.. The Detection Mix consists of 1X Stop & Detect Buffer B, 40 μg/mL ADP Ab, and 4 nM 633 tracer. Assay was inhibited with Staurosporine for 30 minutes prior to the enzymatic reaction at room temperature. 

Dose-Response (Staurosporine AM-2282)

RIPK2 Dose Response Curve

IC50 = 5.63 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 Readout for RIPK2 Titration

FI Detection Mix (10 μL): 1x Stop & Detect Buffer B, 40 μg/mL ADP Ab-IRDye QC1, 8 nM AlexaFluor 594 Tracer. 

TR-FRET

RIPK2 Assay TR-FRET Titration

TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer  C, 8 nM ADP Ab-Tb, 1000 nM ADP HiLyte647 tracer.

RIPK2 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 RIPK2 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 RIPK2 As a Therapeutic Target

RIPK2 plays an important role in regulating inflammatory signaling and cell-death pathways. NOD1 and NOD2 are pattern-recognition receptors that signal to RIPK2. Upon its recruitment by NOD1/2, RIPK2 activates NF-κB and MAPKs, producing pro-inflammatory cytokines (He & Wang 2018). 

Overactivation of NOD1/2 can lead to autoinflammatory diseases, and RIPK2 inhibitors were found to significantly inhibit the immune response of these proteins. Recent studies also show involvement of RIPK2 in the occurrence and progression of several cancers, such as gastric cancer, breast cancer, and renal cell carcinoma (Song et al, 2022).

The Transcreener ADP RIPK2 Assay is an excellent tool for researchers examining the therapeutic effects of RIPK2. 

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