Learn More About Transcreener Method Used for this RIPK1 Assay.

RIPK1 Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Assay directly measures ADP produced by RIPK1  (Receptor-Interacting Serine/Threonine Protein Kinases). These ADP measurements allow researchers to effectively determine the activity of the RIPK1 enzyme. The assay provides a powerful tool to screen compound libraries for RIPK1 modulators to help find new therapies for disease.

The kit comes complete with the detection reagents required to measure activity. RIPK1 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

How Does This RIPK1 Assay Work?

The Transcreener RIPK1 Assay determines RIPK1 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 RIPK1 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 RIPK1 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 RIPK1 Enzymatic Activity

Fluorescence Polarization (FP)

Transcreener RIPK1 Assay Schematic

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

Fluorescence 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 RIPK1
  • Screen Compound Libraries for RIPK1 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 RIPK1 assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized RIPK1 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 = 0.78.

RIPK1 Assay Z' Data

Detection of ADP Under RIPK1 Initial Velocity (FP Readout)

The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 100 μM ATP, 40 mM Tris (pH 7.5), 5 mM MnCl2, and 40 mM MgCl2.  The enzyme buffer includes 40 mM Tris (pH 7.5), 0.1 mM DTT, and 0.2 mg/mL BSA. The Detection mix included 40 μg/mL ADP2 antibody, 4 nM ADP Alexa 633 tracer, and 1X Stop & Detect Buffer B in 10 μL. The RIPK1 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.

RIPK1 Enzyme Titration

RIPK1 Assay Enzyme Titration

Linear Response

RIPK1 Linear Response Curve

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 15.34 μg/mL RIPK1, 40 nM Tris, 0.1 DTT mM, 0.2 mg/mL BSA, 100 μM ATP, 5 mM MnCl2, and 40 mM MgCl2 . 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)

IC50 = 0.4 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

RIPK1 Titration with FI Readout

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

TR-FRET

RIPK1 Titration with TR-FRET Readout

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

RIPK1 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 RIPK1 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
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/iD5ValidatedClick Me ArrowValidatedClick 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 RIPK1 As a Therapeutic Target

Studies show RIPK1 inhibitors have potential therapeutic effects particularly in diseases with atypical tumor necrosis factor receptor (TNFR) signaling (Finger et al, 2017). In necroptosis, the necrosome, a complex of multiple proteins, recruits mixed kinase domain like protein (MLKL), rupturing the cell membrane. RIPK1 has a hand in initiating this. When TNF binds to its receptor, proteins including RIPK1 form TNFR-1 complex I (Mason et al, 2017). Eventually, the signaling pathway requires RIPK1 and RIPK3 to autophosphorylate each other; forming the necrosome.

A team of scientists found that specifically inactivating RIPK1 was superior to eliminating kidney ischemia–reperfusion injury, systemic inflammation compared to the loss of MLKL (Newton et al, 2016). It’s not only inflammatory diseases, but other diseases such as Alzheimer’s disease (AD) could benefit from RIPK1 inhibitors. An overabundance of the protein has been found in patients with AD (Mason et al, 2017).

The Transcreener ADP RIPK1 Assay is an excellent tool for researchers examining the therapeutic effects of RIPK1. 

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