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Application Note: Detection of SARM1 Activity with the Transcreener ADPR Assay

SARM1 Assay - A Transcreener ADPR Assay Application

The Transcreener ADPR Assay, a far-red, competitive immunoassay, detects ADPR produced as SARM1 catalyzes NAD cleavage. SARM1 (Sterile Alfa and TIR Motif Containing 1) plays a critical role in axonal degeneration and is a vital therapeutic target for neurodegenerative disorders. Leverage the trusted Transcreener technology to discover novel SARM1 modulators.

ADPR produced by SARM1 is converted to AMP by a coupling enzyme in real-time. AMP displaces the AMP2/GMP2 Tracer from the highly specific AMP2/GMP2 antibody, resulting in a change in fluorescence. The assay is available in an easy-to-use, HTS-ready format with an FP readout.

The kit comes complete with the detection reagents required to measure activity. SARM1 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.

Sensitive Detection of ADPR to Measure SARM1 Activity

Fluorescent Polarization (FP)

Schematic of SARM1 Assay

Applications

  • Measure Enzymatic Activity of SARM1
  • Screen Compound Libraries for SARM1 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

Features

  • Easy to use, mix-&-read, homogenous format
  • Sensitive Detection of ADPR from 10 nM - 50 μM
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method

Easy-to-Use, Mix-&-Read Format

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

Overview of SARM1 Protocol

Robust Assay Perfect for High Throughput

Robust data (Z' > 0.7) is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z' shown here = 0.85.

Robust Z' Data for SARM1

SARM1 Enzyme Titration Within Initial Velocity Region

Performing the SARM1 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 ADPR Assay. The assay show linearity when raw data is converted to ADPR using a standard curve. Here we performed a 12 point, 2-fold dilution starting at 2 μM SARM1 enzyme. The buffer included 50 mM Tris pH 7.5, 5 mM MgCl2, and 0.01% Triton X-100. The detection mixture included 1X Stop and Detect Buffer, 15 μg/mL AMP2/GMP2 Antibody, and 8 nM AMP2/GMP2 Tracer. The SARM1 enzyme reaction took place for 60 minutes at 30ºC.

SARM1 Enzyme Titration

SARM1 Enzyme Titration in FP Readout

Linear Response

SARM1 Assay Linear Resposne Curve

Discover Novel SARM1 Modulators

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 70 nM SARM1 enzyme, 20 μM NAD+, 50 mM Tris (pH 7.5), 5 mM MgCl2, 0.01% Triton X-100, and 1X ADPR-AMP Coupling enzyme. The Detection Mix included 15 μg/mL AMP2/GMP2 Antibody, 8 nM AMP2/GMP2 Tracer, and 1X Stop and Detect Buffer.

Dose-Response With Inhibitor DSRM-3716

SARM1 Assay Inhibitor Dose Response with DSRM-3716

IC50 = 0.45 μM

SARM1 Assay Services

BellBrook Labs offers Lead Discovery Services for SARM1 using our trusted Transcreener ADPR Assay. BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your program. We 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

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Far-Red FP Readouts Validated on Major Multimode Readers

SupplierInstrumentFP Assays
TriStar2S LB 942In Review
Tristar 5In Review
Mithras2 LB 943In Review
CytationTM 5Validated
CytationTM 3Validated
CytationTM 1Validated
SynergyTM H1Validated
SynergyTM 2/H4/4Click Me ArrowValidated
SynergyTM HTXNot Capable
SynergyTM Neo 2Validated
POLARstar® OmegaClick Me ArrowValidated
FLUOstar® OmegaNot Capable
PHERAstar® FSXClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidated
SenseIn Review
Analyst® GT/HTClick Me ArrowValidated
Gemini® XPS/EMNot Capable
SpectraMax® M2/M2eNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not Capable
SpectraMax® ParadigmClick Me ArrowValidated
SpectraMax® iD3/iD5Click Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidated
Infinite® M200Not Capable
Infinite® F500Click Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidated

What's Included

What You Will Need

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