The Transcreener ENPP1 Assay determines ENPP1 enzyme activity by directly measuring the AMP/GMP formed by the enzyme using the Transcreener AMP2/GMP2 Assay. By detecting AMP/GMP output, the assay provides a universal method to assess the activity of any AMP/GMP producing enzyme in real-time.
Transcreener ENPP1 Assay technology uses a simple but highly effective method that consists of an antibody selective to AMP/GMP and a far-red fluorescent tracer. AMP/GMP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The ENPP1 assay is available with FP 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.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The ENPP1 assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized ENPP1 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 = 0.72
ENPP1 was titrated in the presence of 10 μM ATP or cGAMP in buffer containing 50 mM Tris, 5 mM MgCl2, 1% DMSO, and 0.01% Brij (pH 7.5). The plate was mixed well and incubated for 60 minutes. The enzyme reaction was quenched with an equal volume of EDTA containing stop buffer with 4 nM tracer and 10 μg/mL antibody. The plate was mixed well and read in PHERAstar Plus. Raw data and polarization values were converted into AMP demonstrating initial velocity conditions. The EC50 using cGAMP as a substrate was 48.12 pM. The EC50 using ATP as a substrate was 46.47 pM.
ENPP1 (500 pM) was used to determine the substrate Km values in reactions containing varying amounts of ATP or cGAMP. Staggered incubation periods of 5, 10, 20, 30, 45, and 60 min were used prior to addition of Stop and Detect Mix to allow for initial velocity measurements at all substrate concentrations. Raw data (millipolarization units for FP) were converted to AMP using separate standard curves for each ATP or cGAMP concentration. The initial velocity from each of those time points was fitted to a Michaelis–Menten curve using GraphPad Prism to calculate the apparent Km values.
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 ENPP1 program.
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.
| Component | Notes |
|---|---|
| AMP2/GMP2 Antibody | Antibody specific to the nucleotides AMP and GMP. |
| AMP2/GMP2 Tracer | When displaced, changes in fluorescence can be read in an FP or TR-FRET readout, depending on the assay format. |
| Tris Solution | Used to buffer the detection mixture. |
| AMP and GMP | AMP and GMP are used to create the ATP/AMP or cGAMP/AMP & GMP standard curve. |
| Component | Notes |
|---|---|
| ENPP1 Enzyme | We have successfully used recombinant ENPP1 from R&D Systems Cat. #6136. |
| Enzyme Buffer | Enzyme buffer components optimized for your target. See the technical manual for potential interfering components. |
| ATP or cGAMP | Use the nucleotide required for your experiment. It is possible to purchase nucleotides direct from BellBrook Labs. |
| Stop Buffer B, 5X | Buffer containing EDTA capable of stopping the ENPP1 reaction. If you'd like to use this please mention it in your order and it will be included from BellBrook Labs Cat. #2139 - 1 mL, Cat. #2140 - 10 mL. |
| 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 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 AMP/GMP 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. |
There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.
In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets. The enzyme targets discussed include:
ENPP1 is a nucleotide pyrophosphatase and phosphodiesterase that regulates purinergic signaling and can degrade different nucleotides including ATP and cGAMP. ENPP1 has been identified as the primary enzyme responsible for degrading cGAMP and therefore is under intense investigation as a therapeutic target for cancer immunotherapy.
The Transcreener ENPP1 Assay is an excellent tool for researchers examining the therapeutic effects of ENPP1.
Find the right resources to help you get to your solution faster.
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.
BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
