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.
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.
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.
Performing the SARM1 titration within the initial velocity region (<20% conversion of substrate to product) confirms that the signal is within dynamic range for optimized transcreener adpr assay. assay show linearity when raw data converted using a standard curve. here we performed 12 point, 2-fold dilution starting at 2 μm sarm1 enzyme. buffer included 50 mm tris ph 7.5, 5 mgcl2, and 0.01% triton x-100. detection mixture 1x stop 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.
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.
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.
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| Component | Notes |
|---|---|
| AMP2/GMP2 Antibody | Sufficient antibody is included in the kit to complete 1,000 assays or 10,000 assays. The final antibody concentration in the 20 μL complete assay is 7.5 μg/mL. |
| AMP2/GMP2 AlexaFluor 633 Tracer | AlexaFluor® 633 labeled tracer. The final tracer concentration in the 20 μL complete assay is 4 nM. When displaced, changes in fluorescence can be read in an FP format. |
| ADPR-AMP Coupling Enzyme | Coupling enzyme in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, 10% glycerol. |
| Stop & Detect Buffer B, 10X | The Stop & Detect Buffer B will stop the coupling enzyme by chelating Mg2+. To ensure that the reaction is completed stopped, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction. The final concentration of Stop & Detect Buffer B in the complete 20 μL reaction is 0.5X. |
| ADPR | The ADPR in this kit can be used to create a standard curve to convert mP values to ADPR product formed. |
| Component | Notes |
|---|---|
| SARM1 Enzyme | We have successfully used recombinant human SARM1 protein from BPS Bioscience Cat. #100069. A SARM1 concentration of 70 nM was used for screening and SAR. |
| Enzyme Buffer | 50 mM Tris (pH 7.5), 5 mM MgCl2,, and 0.01% Triton X-100. Enzyme buffer components may be optimized as required. |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the AMP 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
