Nov 07, 2025

Public workspaceTranscreener® OAS FP Assay Technical Manual

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx449ol8j/v1
  • info 1
  • 1BellBrook Labs LLC
info info
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.kxygx449ol8j/v1
Protocol Citation: info 2025. Transcreener® OAS FP Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx449ol8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 22, 2025
Last Modified: November 07, 2025
Protocol Integer ID: 230532
Keywords: transcreener oas fp assay, transcreener assay, transcreener amp2 assay, simple biochemical assay, oas2, assay, such as oas1, enzyme, turn inhibition of protein synthesis, oas activity, host rna, measuring oas activity, oas3, stranded rna, oas1, invading rna virus, rna virus, oa, cell from viral infection, 5a from atp, dsrna, signaling event, competitive fluorescence polarization, coupling enzyme, high throughput screening, protein synthesis, family of enzyme, presence of viral invader, innate immune system
Abstract
The Transcreener® OAS FP Assay is a far-red, competitive fluorescence polarization (FP) assay (Figure 1). The assay is designed to be used with the 2’-5’-oligoadenylate synthase (OAS) family of enzymes such as OAS1, OAS2, and OAS3), that produce the product 2-5A. The Transcreener OAS FP Assay is simple biochemical assay for measuring OAS activity based on the Transcreener AMP2 Assay. The assay uses a coupling enzyme to convert 2-5A into AMP in the presence of ATP and dsRNA.
Double stranded RNA enters the cell from viral infection. As a first line of defense in the innate immune system, OAS is activated by viral double-stranded RNA. Upon activation, OAS synthesizes 2-5A from ATP which in turn activates RNase L. The result of this signaling event cleaves both viral and host RNA and in turn inhibition of protein synthesis in the cell, thus ending the replication of invading RNA viruses. Mutation or inadvertent stimulation can produce autoimmune conditions by activating the pathway inconsistently even while not in the presence of viral invaders.
The Transcreener assay is designed specifically for high throughput screening (HTS), with a single-addition, mix-and-read format. It offers reagent stability and compatibility with commonly used multimode plate readers.
Troubleshooting
Introduction
The Transcreener® OAS FP Assay is a far-red, competitive fluorescence polarization (FP) assay (Figure 1). The assay is designed to be used with the 2’-5’-oligoadenylate synthase (OAS) family of enzymes such as OAS1, OAS2, and OAS3), that produce the product 2-5A. The Transcreener OAS FP Assay is simple biochemical assay for measuring OAS activity based on the Transcreener AMP2 Assay. The assay uses a coupling enzyme to convert 2-5A into AMP in the presence of ATP and dsRNA.
Double stranded RNA enters the cell from viral infection. As a first line of defense in the innate immune system, OAS is activated by viral double-stranded RNA. Upon activation, OAS synthesizes 2-5A from ATP which in turn activates RNase L. The result of this signaling event cleaves both viral and host RNA and in turn inhibition of protein synthesis in the cell, thus ending the replication of invading RNA viruses. Mutation or inadvertent stimulation can produce autoimmune conditions by activating the pathway inconsistently even while not in the presence of viral invaders.
The Transcreener assay is designed specifically for high throughput screening (HTS), with a single-addition, mix-and-read format. It offers reagent stability and compatibility with commonly used multimode plate readers.
The Transcreener OAS FP Assay provides the following benefits:
  • A simple single addition OAS activity assay capable of HTS.
  • Excellent data quality (Z’ ≥ 0.7) and signal (≥100 mP polarization shift) for determining OAS activity by measuring 2-5A production.
  • Far-red tracer further minimizes interference from fluorescent compounds and light scattering.

Figure 1. Schematic overview of the Transcreener OAS FP Assay. The Transcreener 2-5A Detection Mixture contains a coupling enzyme that generates AMP and ATP from 2-5A, and an AMP AlexaFluor® 633 tracer bound to an AMP antibody. AMP produced by the coupling enzyme displaces the tracer, which rotates freely, causing a decrease in FP. Note that the coupling enzyme regenerates ATP, preventing OAS substrate depletion.

Product Specification

ProductQuantityPart #
Transcreener® OAS FP Assay1,000 assays*3027-1K
10,000 assays*3027-10K
*The exact number of assays depends on enzyme reaction conditions. The kits are designed for use with 384-well plates, using 20 µL reaction volumes.

IMPORTANT: Antibody centrifugation is required to remove aggregates that can disrupt data quality. Antibodies should be centrifuged at 10,000 x g for 10 minutes before use. Following centrifugation, pipet the solution needed from the top of the aliquot to ensure precipitate is not present in the detection reagents.
Storage Store all reagents at –20°C upon receipt. Please recommend avoiding freeze thaw cycles for the best result. Please aliquot and store if not using multiple reagents at one time. A maximum of 3 freeze-thaw cycles have shown no effect on assay results.
Use the reagents provided in this kit within 1 year from date of receipt.
Materials Provided

ComponentCompositionNotes
AMP2/GMP2 Antibody1.26 mg/mL solution in PBS with 10% glycerol*Sufficient antibody is included in the kit to complete 1,000 assays (Part # 3027-1K) or 10,000 assays (Part # 3027-10K).
AMP2/GMP2 Alexa Fluor® 633 Tracer800 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35The final tracer concentration in the 20 µL reaction is 4 nM. Sufficient tracer is included in the kit to complete 1,000 assays (Part # 3027-1K) or 10,000 assays (Part # 3027-10K).
2-5A–AMP Coupling Enzyme1000 nM 2-5A-AMP Coupling Enzyme in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, 25% glycerolSufficient coupling enzyme present in excess to ensure 2-5A is converted to AMP for detection by detection reagents.
ATP5 mM ATP in deionized water, pH 7.0The ATP is the substrate for OAS and is regenerated by the coupling enzyme in the detection mixture
AMP5 mM AMP in deionized water, pH 7.0AMP is used to create a standard curve.
dsRNA10 mg/mL dsRNA in RNAse free water.dsRNA is required to activate OAS enzymes.
OAS Buffer, 10X250 mM Tris (pH7.5), 100 mM MgCl2 and 0.1% Brij-35Buffer prepared for use with OAS enzymes. Buffer changes may be necessary depending on experimental design.
*The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.

Materials Required but Not Provided
  • 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.
  • Enzyme—Transcreener OAS assays are designed for use with purified OAS enzyme preparations. Contaminating enzymes, such as phosphatases or nucleotidases, can produce background signal and reduce the assay window.
  • Plate Reader—A multidetection microplate reader configured to measure FP of the AMP Alexa Fluor 633 tracer is required. Transcreener FP Assays have been successfully used on the following instruments: BioTek Synergy™2 and Synergy™4; BMG Labtech PHERAstar Plus and CLARIOstar Plus; Molecular Devices SpectraMax™ Paradigm; Perkin Elmer EnVision and ViewLux; and Tecan Infinite F500, Safire 2™, and M1000.
  • Assay Plates—It is important to use assay plates that are entirely black with a nonbinding surface. We recommend Corning® 384-well plates (Cat. # 4514). The suggested plate has a square well top that enables easier robotic pipetting and a round bottom that allows good Z’ factors. It has a recommended working volume of 15–20 µL.
  • Liquid Handling Devices—Use liquid handling devices that can accurately dispense a minimum volume of 2.5 µL into 384-well plates.

Note
Note: Contact BellBrook Labs Technical Service for suppliers and catalog numbers for buffer components, and additional information regarding setup of FP instruments.

Before You Begin
1. Read the entire protocol and note any reagents or equipment needed (see Section 2.2). 2. Check the FP instrument and verify that it is compatible with the assay being performed (see Section 4.1).
Protocol
The Transcreener OAS FP Assay protocol consists of 3 steps (Figure 2). The protocol was developed for a 384-well format, using a 10 µL enzyme reaction and 20 µL final volume when the plates are read. The use of different densities or reaction volumes will require changes in reagent quantities (see Section 7.2 for example reaction volumes). Once the instrument parameters and enzyme optimization are complete, the assay itself consists of a single step - simply add detection reagents to your enzyme reaction and read the plate.

Figure 2. An outline of the procedure. The procedure consists of 3 main steps with a mix-and-read assay format.

Set Up the Instrument
Becoming familiar with ideal instrument settings for FP is essential to the success of the Transcreener OAS FP Assay.

Note
Note: A complete list of instruments and instrument-specific application notes can be found online at: https://www.bellbrooklabs.com/technicalresources/instrument-compatibility
Contact BellBrook Labs Technical Service if you have questions about settings and filter sets for a specific instrument.
4.1.1 Verify That the Instrument Measures FP Ensure that the instrument is capable of measuring FP (not simply fluorescence intensity) of AMP AlexaFluor 633 Tracer.
4.1.2 Define the Maximum mP Window for the Instrument
Measuring high (tracer + antibody) and low (free tracer) FP will define the maximum assay window of your specific instrument. Prepare High and Low FP Mixtures in quantities sufficient to perform at least 6 replicates for each condition.
Use AMP Alexa Fluor 633 Tracer at 4 nM in your enzyme buffer in a 20 µL complete assay. This mimics the 2-fold dilution when adding an equal volume of detection mixture to an enzyme reaction. As an example, the 1X AMP Detection Mixture may contain 8 nM tracer. After adding this to the enzyme reaction, the concentration in the final 0.5X, 20 µL complete assay would be 4 nM.
High FP Mixture Prepare the following solution.

ComponentStock ConcentrationComplete Assay ConcentrationExample: 25 AssaysYour Numbers
AMP2 Antibody1.26 mg/mL*16.0 µg/mL6.3 µL**
AMP Alexa Flour 633 Tracer 800 nM4 nM2.5 µL
OAS Enzyme Buffer, 10X10 X1 X50.0 µL
Water441.2 µL
Total500.0 µL
*Please note AMP2 Antibody concentration varies by lot number. This is an example and should be adjusted based on stock concentration accordingly.
**Pipetting small sample volumes accurately requires the correct equipment and proper technique. An extra dilution step may be required to ensure accuracy.

Note
Note: The complete assay concentrations with the Stop & Detect Buffers are based on a 20 μL final volume.

Low FP Mixture Prepare the following solution.

ComponentStock ConcentrationComplete Assay ConcentrationExample: 25 AssaysYour Numbers
AMP Alexa Fluor 633 Tracer800 nM4 nM2.5 µL
OAS Enzyme Buffer, 10X1 X50.0 µL
Water447.5 µL
Total500.0 µL

4.1.3 Measure the FP Subtract the Low FP Mixture readings from the corresponding High FP Mixture readings. The difference between the low and high FP values should be >100 mP.

Note
Caution: Contact BellBrook Labs Technical Service for assistance if the assay window is <100 mP.

Optimize the Enzyme Concentration
Perform an enzyme titration to identify the optimal enzyme concentration for the Transcreener OAS FP Assay. Use enzyme buffer conditions and substrate concentrations that are optimal for your enzyme and experimental goals. If a compound screen is planned, you should include the library solvent at its final assay concentration. Run your enzymatic reaction at its requisite temperature and time period.
4.2.1 Enzyme Titration Steps For a robust assay, it is recommended to use an OAS concentration that produces at least a 100 mP shift (see Figure 3). This is usually achieved using an enzyme concentration that results in 50-80% of the maximum polarization shift (EC50 to EC80). To determine the EC80 enzyme concentration, use the following equation:

ECX = (X ÷ (100 – X) )(1 ÷ |hillslope| ) × EC50


Figure 3. Enzyme titration curve. Titration with the EC80 concentration indicated. The EC80 may vary based on enzyme lot. Please use C of A for the recommended EC80 for your assay.

4.2.2 Enzyme Assay Controls The enzyme reaction controls define the limits of the enzyme assay

ComponentNotes
0% 2-5A Conversion ControlThis control consists of the 2-5A Detection Mixture, the enzyme reaction components (without OAS enzyme, 0 mM AMP). It defines the upper limit of the assay window.
100% 2-5A Conversion ControlThis control consists of the ADP Detection Mixture, the enzyme reaction components (without OAS enzyme, 1 mM AMP). It defines the lower limit of the assay window.
Minus-Nucleotide ControlTo verify that the enzyme does not interfere with the detection module, perform an enzyme titration in the absence of ATP.
ATP/AMP Standard CurveAlthough optional, a standard curve can be useful to ensure day-to-day reproducibility and that assay conditions were performed using initial rates. It can also be used to calculate product formed. See Section 7.1 for a description of how to run the standard curve.
Background ControlUse only enzyme reaction conditions including 1X OAS buffer.

Run an Assay
4.3.1 Experimental Samples 1. Add the enzyme to the test compounds at the desired concentration. The total volume of this mixture is 5 µL. Mix on a plate shaker. Incubate the enzyme inhibitor mixture for the desired time (typically at least 30 minutes). 1. Prepare both the substrates and 1X 2-5A Detection Mixture prior to starting the enzymatic assay. Since the assay is to be run in continuous mode, we recommend the 2-5A Detection Mixture will be added directly after the substrates in order to effectively measure enzymatic activity. 2. Start the enzyme reaction by adding 5 µL of ATP and dsRNA, then mix. It is recommended to use concentrations of 1 mM ATP and 10 µg/mL dsRNA, in the 10 µL enzyme reaction mixture. Concentrations may vary based on your experiment. Note: The final volume of the enzyme reaction mixture should be 10 µL for 384 well plates. Use 2X ATP (2 mM) and 2X dsRNA (20 µg/mL), in 5 µL to achieve the appropriate final concentration. See Section 7.2 for a list of other plate formats 3. Prepare 1X 2-5A Detection Mixture as follows: The detection mixture should be made as fresh as possible. It has a deck stability up to 2 hours after being prepared.



1X 2-5A Detection Mixture
ComponentStockDetection Mix Conc.Example VolumeYour Numbers
AMP2 Antibody1.26 mg/mL32.0 µg/mL254 µL
AMP Alexa Fluor 633 Tracer800 nM8 nM100 µL
Coupling Enzyme1,000 nM62.5 nM 625 µL
OAS Enzyme Buffer10X1X1,000 µL
Water8,021 µL
Total10,000 µL
Note: This is an example of running an assay for HTS or to obtain a dose response. Your volumes and concentrations may vary. It is important to have a 1:1 ratio of enzyme mix and detection mix for the final assay readout.

4. Add 10 µL of 1X 2-5A Detection Mixture to 10 µL of the enzyme reaction. Mix using a plate shaker. Note: After detection mixture is added to enzyme reaction the final concentration of components in a 20 µL complete assay will be 0.5X the Detection Mixture (4 nM tracer, 16 µg/mL AMP2 Antibody 31.25 nM Coupling Enzyme, and 0.5 mM ATP). 5. Mix gently on a plate shaker(~1 minute). Incubate at 30°C for 1 hour and measure FP


4.3.2 2-5A Detection Controls These controls are used to calibrate the FP plate reader and are added to wells that do not contain enzyme.
ComponentNotes
Minus Antibody (Free Tracer) ControlThis control contains the AMP Tracer without the AMP2 Antibody and is set to low mP, typically between 20-50 mP depending on the instrument.
Minus Tracer ControlThis control contains the AMP2 Antibody without the AMP Tracer and is used as a sample blank for all wells. It contains the same AMP2 16 μg/mL Antibody concentration in all wells.

General Considerations

Continuous Mode Assay
The Transcreener OAS Assay Kit does not have a stop solution currently available. The assay has to be performed in continuous mode to determine enzymatic activity. The equilibration time for the tracer and AMP2 Antibody can be greater than 15 minutes, making it difficult to quantitate AMP produced during short-term enzyme reactions. We recommend reading the plate after 60 minutes for the best results.
Reagent and Signal Stability
5.2.1 Signal Stability Since the assay is a continuous mode the signal will change as the enzyme produces more product. We recommend reading the assay at 60 minutes after addition of the 2-5A Detection Mixture for the best results. It is important to read plates at the same interval to avoid plate to plate variability when performing high throughput screens.
5.2.2 2-5A Detection Mixture Stability The 2-5A Detection Mixture is stable for up to 2 hours at room temperature (20–25°C) before addition to the enzyme reaction (i.e., when stored on the liquid handling deck).
Frequently Asked Questions

QuestionPossible Solutions
Other Transcreener Assays require adjustment of antibody concentration. Is that something I need to do for the OAS FP Assay?Unlike other Transcreener assays, the OAS FP Assay does not require adjustment to the antibody concentration. It is designed to be used within the working range of initial velocity for OAS enzymes.
No change in FP observedLow antibody/tracer activity or Δ mP signal.
  • The tracer and antibody are stable for up to 6 freeze-thaw cycles. For frequent use aliquot the antibody and tracer and store the aliquots at -20°C. Use a minimum of 20 μL aliquots.
  • Other components of the detection mix should not have multiple freeze-thaw cycles. Aliquot reagents and store at -20°C for future use.
Is a standard curve required?No, it is not required to run a standard curve. We recommend running the ATP/AMP standard curve, if you want to convert raw mP values to product formed. While designing a standard curve, make sure that most of the points are between 0% and 30% conversion (initial velocity). We do not recommend using a standard curve from previous experiments, rather generate a new curve with each experiment to achieve the most accurate result.
Can this assay be used with cell lysates?The assay will only work with purified recombinant OAS protein. The presence of nucleases in the lysates prohibits the use of Transcreener assays with lysates.
High background signal or change in signal after incubation with detection mixture.
  • Buffers should be made fresh with nuclease free water to prevent non-specific hydrolysis of nucleotides.
  • Be sure to not leave the plate in the instrument between the reads as this may cause evaporation leading to high background.
  • Use non-binding black plates. Medium binding or high binding plates will cause increased assay variability.
Why is my window with OAS very small?Example reasons for a diminished window
  • Be sure that OAS enzyme has activity. We recommend using OAS from a commercial source as a positive control as needed.
  • Handle the coupling enzyme with care. We recommend storing the enzyme at -20°C and aliquoting to prevent loss of activity.
  • Since the equilibration time of the assay is 10-15 minutes, start reading the plate at 60 min. Earlier time points may lead to diminished signal.

Appendix

Standard Curve
The standard curve mimics any enzyme reaction where 2-5A is formed and then converted to AMP. In this example a 12-point standard curve was prepared using the concentration of ATP and AMP show in Table 1. Commonly, 8-12 point standard curves are used. In this case you may omit ATP or not run a standard curve depending on your experiment.

% ConversionAMP (µM)ATP (µM)
1001000.00
5.0050.0950.0
2.5025.0975.0
1.2512.5987.5
1.0010.0990.0
0.505.0995.0
0.303.0997.0
0.202.0998.0
0.101.0999.0
0.050.5999.5
0.020.2999.8
0.010.1999.9
001000.0
Table 1. AMP Standard Curve. Standard curve to help convert raw mP values to product formed.

Use the following equations to calculate the Z’ factor:



Using the Assay with Different Volumes and Plate Formats

ComponentTotal VolumeEnzyme Reaction Volume2-5A Detection Mix Volume
96 Well Low Volume Plate50 µL25 µL25 µL
384 Well Low Volume Plate20 µL10 µL10 µL
1536 Well Low Volume Plate8 µL4 µL4 µL
Please check the working plate volumes from the manufacturer to ensure they are within the suggest volumes ranges of your plate.

Summary of Additive Effects on the Transcreener OAS FP Assay
The assay window was determined to have limited effect with certain components when used under the recommended conditions. To determine the additive affects of a buffer component please test by titrating the component in the known concentration range. You can use only the detection mix and a standard curve to determine the effect on assay performance, however we would suggest use of the OAS enzyme to understand effects on the assay system with OAS enzyme. Below are common components used in enzyme buffers. The maximum tolerance is defined as less than 10% drop in mP observed at the listed concentration below. Tolerance listed includes human OAS1 enzyme.

ComponentMaximum Tolerance
DMSO10%
MeOH1.25%
Brij-350.25%
Triton X-1000.03%
EDTA1.25%
1Mg Cl26.25 mM
NaCl12.5 mM
Imidazole25 mM
BSA0.125 mg/mL
BGG0.125 mg/mL

Contact Information
Email: [email protected] Phone: 608.443.2400 Toll-Free: 866.313.7881 FAX: 608.441.2967