Oct 31, 2025

Public workspaceTranscreener® AMP2/GMP2 TR-FRET Assay Technical Manual

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg31bqpl25/v1
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg31bqpl25/v1
Protocol Citation: info 2025. Transcreener® AMP2/GMP2 TR-FRET Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31bqpl25/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 02, 2025
Last Modified: October 31, 2025
Protocol Integer ID: 228868
Keywords: amp2/gmp2 tr-fret assay, assay development for new hts target, assay development, inhibitor profiling, inhibitor profiling across multiple target family, substrate nucleotide, competitive immunoassay for enzyme, selectivity over substrate nucleotide, assay, specific monoclonal antibody, enzyme, new hts target, phosphodiesterase, protein ligase, throughput screening, gmp, competitive immunoassay, nucleic acid, including atp
Abstract
The Transcreener® AMP2/GMP2 TR-FRET Red Assay is a competitive immunoassay for enzymes that produce AMP or GMP with a far-red, time-resolved Förster-resonance-energy-transfer (TR-FRET) readout. The assay relies on a highly specific monoclonal antibody that recognizes AMP or GMP with more than 1,000-fold selectivity over substrate nucleotides, including ATP, cAMP, or cGMP. Enzymes that can be used with the assay include ubiquitin, SUMO, nucleic acid and protein ligases, phosphodiesterases (PDEs), and synthetases.
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 generic nature of the Transcreener® HTS assay platform eliminates delays involved in assay development for new HTS targets and greatly simplifies compound and inhibitor profiling across multiple target families.
Troubleshooting
Introduction
The Transcreener® AMP2/GMP2 TR-FRET Red Assay is a competitive immunoassay for enzymes that produce AMP or GMP with a far-red, time-resolved Förster-resonance-energy-transfer (TR-FRET) readout. The assay relies on a highly specific monoclonal antibody that recognizes AMP or GMP with more than 1,000-fold selectivity over substrate nucleotides, including ATP, cAMP, or cGMP. Enzymes that can be used with the assay include ubiquitin, SUMO, nucleic acid and protein ligases, phosphodiesterases (PDEs), and synthetases.
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 generic nature of the Transcreener® HTS assay platform eliminates delays involved in assay development for new HTS targets and greatly simplifies compound and inhibitor profiling across multiple target families.
The Transcreener® AMP2/GMP2 TR-FRET Red Assay provides the following benefits:
  • Accommodates substrate concentrations ranging from 0.1 µM to 1,000 µ
  • Excellent data quality (Z’ ≥ 0.7) at low substrate conversion (typically 10–30%).
  • The only assay method that allows detection of unmodified AMP and GMP without using coupling enzymes.
  • Overcomes the need for time-consuming, one-off assay development for individual members within a group transfer enzyme family by using a single set of assay reagents that detect an invariant product.
  • Time-gated detection method largely eliminates interference that can result from prompt fluorescence of test compounds.
  • Far-red tracer further minimizes interference from fluorescent compounds and light scattering.


Figure 1. Schematic overview of the Transcreener AMP2/GMP2 TR-FRET Red Assay. The Transcreener® AMP/GMP Detection Mixture contains an AMP/GMP HiLyte647 tracer bound to an AMP2/GMP2 antibody conjugated to terbium (Tb). Excitation of the Tb complex in the UV range (~330 nm) results in energy transfer to the tracer and emission at a higher wavelength (665nm) after a time delay. AMP/GMP produced by the target enzyme displaces the tracer, which causes a decrease in TR-FRET.

Product Specifications

ProductQuantityPart #
Transcreener® AMP2/GMP2 TR-FRET Red Assay1,000 assays*3020-1K
10,000 assays*3020-10K
*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 -20ºC upon receipt.
Materials Provided

ComponentCompositionNotes
AMP/GMP HiLyte647 Tracer10 µM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35The concentration of AMP/GMP HiLyte647 Tracer needed for an enzyme target depends upon the initial ATP/cAMP/cGMP concentration and buffer conditions in the enzyme reaction (see Section 4.2). Sufficient tracer is included in the kit to complete 1,000 assays (Part # 3021-1K) or 10,000 assays (Part # 3021-10K) at an ATP/cAMP/GMP concentration up to 100 µM.
AMP2/GMP2 Antibody-Terbium Conjugate800 nM solution in HEPES-buffered salineThe final antibody concentration in the reaction is 4 nM in a 20 µL final reaction volume.
Tris Solution1 M Tris (pH 7.5)Tris Solution is used to buffer the Detection Mixture.
AMP5 mMAMP is used to create an ATP/AMP or cAMP/AMP standard curve.
GMP5 mMGMP is used to create a cGMP/GMP standard curve

Note
Caution: AMP and GMP are common reagents in many laboratories; however, it is imperative that highly purified preparations be used for the Transcreener® assay. If the AMP or GMP stocks contain impurities, the assay window will be compromised.

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® AMP/GMP assays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphatases or nucleotidases, can produce background signal and reduce the assay window.
  • Enzyme Buffer Components—User-supplied enzyme buffer components include enzyme, enzyme cofactors, substrates, and test compounds.
  • Plate Reader—A microplate reader configured to measure TR-FRET of the Tb:HiLyte647 donor:acceptor pair is required. This assay has been designed to provide high-quality data on any HTS-qualified instrument configured to measure TR-FRET using standard europium or terbium complexes with emission wavelengths at 615 nm and 665 nm. Validation was completed using PHERAstar Plus Ex337/Em620/Em665 (BMG LABTECH) and Envision Ex320/ Em615/Em665 (Perkin Elmer).
  • Assay Plates—It is important to use assay plates that are entirely white with a nonbinding surface. We recommend Corning® 384-well plates (Cat. # 4513).
  • 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 TR-FRET instruments.

Before You Begin
1. Read the entire protocol and note any reagents or equipment needed (see Section 2.2). 2. Check the TR-FRET instrument and verify that it is compatible with the assay being performed (see Section 4.1).
Protocol
The Transcreener® AMP2/GMP2 TR-FRET Red Assay protocol consists of 4 steps (Figure 2). The protocol was developed for a 384-well format, using a 15 µL enzyme reaction and 20 µL final volume at the time that the plates are read. The use of different densities or reaction volumes will require changes in reagent quantities.

Figure 2. An outline of the procedure. The assay consists of 4 main steps with a mix-and-read format.

Set Up the Instrument
Becoming familiar with ideal instrument settings for TR-FRET is essential to the success of the Transcreener® AMP2/GMP2 TR-FRET Red Assay.

4.1.1 Verify That the Instrument Measures TR-FRET

Ensure that the instrument is capable of measuring TR-FRET (not simply fluorescence intensity) of the terbium:HiLyte647 TR-FRET pair (Ex320/Em615/Em665).

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.

4.1.2 Define the Maximum TR-FRET Window for the Instrument

Measuring high (0% ATP/cAMP/cGMP conversion) and low (100% ATP/cAMP/cGMP conversion) FRET will define the maximum assay window of your specific instrument. Prepare High and Low FRET Mixtures in quantities sufficient to perform at least 6 replicates for each condition.
Use ATP/cAMP/cGMP at 0.75X and AMP/GMP HiLyte647 Tracer at 0.25X concentration in a 20 µL final reaction volume. This mimics the 2-fold dilution when adding an equal volume of detection mixture to an enzyme reaction. As an example, the 1X detection mixture may contain 10 µM ATP/cAMP/cGMP. After adding this to the enzyme reaction, the concentration in the final 20 µL reaction volume would be 7.5 µM.
High FRET Mixture
Prepare the following solution:

ComponentStock ConcentrationFinal ConcentrationExample: 25 AssaysYour Numbers
AMP2/GMP2 Antibody-Tb800 nM3 nM1.9 µL
Tris Solution1 M18.75 mM9.4 µL
AMP/GMP HiLyte647 Tracer10 µM15 nM0.8 µL
ATP/cAMP/cGMP5 mM7.5 µM0.8 µL
Water487.3 µL
Total500.0 µL
The assay window will depend upon your initial ATP/cAMP/cGMP concentration. These volumes can be adjusted for fewer assays and different ATP/cAMP/cGMP concentrations.

Low FRET Mixture
Prepare the following solution:

ComponentStock ConcentrationFinal ConcentrationExample: 25 AssaysYour Numbers
AMP2/GMP2 Antibody-Tb800 nM3 nM1.9 µL
Tris Solution1 M18.75 mM9.4 µL
AMP/GMP HiLyte647 Tracer10 µM15 nM0.8 µL
AMP/GMP5 mM7.5 µM0.8 µL
Water487.3 µL
Total500.0 µL
The assay window will depend upon your initial AMP/GMP concentration. These volumes can be adjusted for fewer assays and different AMP/GMP concentrations.

4.1.3 Measure the TR-FRET

Test the Z’ factor and assay window on your instrument by adding 20 µL of the Low FRET Mixture in 16 wells and 20 µL of High FRET Mixture in 16 wells. Calculate the Z’ factor using the equation below; values greater than 0.7 are acceptable.


Note
Caution: Contact BellBrook Labs Technical Service for assistance if the calculated Z’ factor is less than 0.7.

Determine the AMP/GMP HiLyte647 Tracer Concentration
The Transcreener® AMP2/GMP2 TR-FRET Red Assay requires detection of AMP/GMP in the presence of excess ATP/cAMP/cGMP (assuming initial velocity enzyme reaction conditions) using an antibody with a finite selectivity for the monophosphate vs. the triphosphate or cyclic monophosphate. The concentration of AMP/GMP HiLyte647 tracer determines the total assay window and the AMP/GMP detection range; the amount needed primarily depends upon the initial ATP/cAMP/cGMP concentration in the enzyme reaction.

Figure 3. Linear relationship between [ATP/cAMP/cGMP] and [AMP/GMP Tracer]. The tracer concentration can be calculated using the equation: y = 3.78x + 22.3

4.2.1 Calculating the Tracer Amount

As shown in Figure 3, the relationship between ATP/cAMP/cGMP and AMP/GMP HiLyte647 Tracer concentrations is linear. (Though shown for up to 100 µM ATP/cAMP/cGMP, the relationship is valid to 1,000 µM.) Therefore, the quantity of AMP/GMP HiLyte647 Tracer for enzyme reactions that use between 0.1 μM and 1,000 μM initial ATP/cAMP/cGMP can be determined using the equation y = mx + b, where x = initial [ATP/cAMP/cGMP] (µM) in the 15 μL enzyme reaction, y = [AMP/GMP HiLyte647 Tracer] (nM) in the 5 μL of 1X AMP/GMP Detection Mixture, m (slope) = 3.78, and b (y-intercept) = 22.3. We recommend a final reaction volume of 20 μL.

For example, if you are using 3 µM ATP/cAMP/cGMP in a 15 µL enzyme reaction, the optimal AMP/GMP HiLyte647 Tracer concentration in the 1X GDP Detection Mixture (assuming 5 µL of AMP/GMP Detection Mixture was added to each 15 µL enzyme reaction) would be (3.78 × 3) + 22.3 = 33.6 nM.


4.2.2 Optimizing the Tracer Concentration

Using the AMP/GMP HiLyte647 Tracer concentration calculated using the equation in Figure 3 will produce excellent results for most users. If it does not produce the results you require, simply optimize the tracer concentration in a stepwise fashion using the AMP/GMP HiLyte647 Tracer concentration (X) from the line as a starting point. Try performing a standard curve (see Section 7.1) at 0.5 × [Y], [Y], and 1.5 × [Y] tracer concentrations to find an assay window that suits your needs. See Section 6 for troubleshooting suggestions.
Optimize the Enzyme Concentration
Perform an enzyme titration to identify the optimal enzyme concentration for the Transcreener® AMP2/GMP2 TR-FRET Red Assay. Use enzyme buffer conditions, substrate, and ATP/cAMP/cGMP concentrations that are optimal for your target enzyme and AMP/GMP HiLyte647 Tracer concentration calculated as described in Section 4.2. 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. Refer to Section 7.2 for the tolerance of different components for your buffer conditions.
4.3.1 Enzyme Titration Steps
To achieve the most robust assay and a high signal, the quantity of enzyme required to produce a 50–80% change in FRET signal is ideal (EC50 to EC80) for screening of large compound libraries and generating inhibitor dose-response curves (see Figure 4). To determine the EC80 enzyme concentration, use the following equation:

EC80 = (80 ÷ (100 – 80) )(1 ÷ hillslope) × EC50

Figure 4. Enzyme titration curve. The ideal range of enzyme concentrations is shown in red.

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

ComponentNotes
0% ATP/cAMP/cGMP Conversion ControlThis control consists of the AMP/GMP Detection Mixture, the enzyme reaction components (without enzyme), and 100% ATP/cAMP/cGMP (0% AMP/GMP). It defines the upper limit of the assay window.
100% ATP/cAMP/cGMP Conversion ControlThis control consists of the AMP/GMP Detection Mixture, the enzyme reaction components (without enzyme), and 100% AMP/GMP (0% ATP/cAMP/cGMP). It defines the lower limit of the assay window.
Minus-Nucleotide Control and Minus-Substrate ControlTo verify that the enzyme does not interfere with the detection module, perform an enzyme titration in the absence of nucleotide (i.e., ATP/cAMP/cGMP).
AMP/GMP Standard CurveAlthough optional, an AMP/GMP standard curve can be useful to ensure day-to-day reproducibility and that the assay conditions were performed using initial rates. It can also be used to calculate product formed and inhibitor IC50 values. See Section 7.1 for a description of how to run the standard curve.
Background ControlUse only 0.5X enzyme reaction conditions and Tris Solution.

Run an Assay
1. Add the enzyme reaction mixture to test compounds and mix on a plate shaker. 2. Start the reaction by adding ATP/cAMP/cGMP, then mix. The final volume of the enzyme reaction mixture should be 15 µL. Incubate at a temperature and time ideal for the enzyme target before adding the AMP/GMP Detection Mixture. 3. Prepare 1X AMP/GMP Detection Mixture as follows:

ATP Concentration: Examples
Component1 µM10 µM100 µMYour Numbers
AMP2/GMP2 Antibody-Tb100 µL100 µL100 µL
AMP/GMP HiLyte647 Tracer13.1 µL30.1 µL200.2 µL
Tris Solution (1 M)125 µL125 µL125 µL
Water4,762.0 µL4745.0 µL4574.8 µL
Total5,000 µL5,000 µL5,000 µL
Final concentrations in the detection mixture should be 16 nM AMP2/GMP2 Antibody-Tb, 25 mM Tris Solution, and the tracer concentration calculated using the equation in Figure 3. An example is shown below:

y=3.78x + 22.3
ABCD
ATP1 µM10 µM100 µM
AMP/GMP HiLyte647 Tracer26.1 nM60.1 nM400.3 nM
4. Add 5 µL of 1X AMP/GMP Detection Mixture to 15 µL of the enzyme reaction. Mix using a plate shaker. 5. Incubate at room temperature (20–25°C) for at least 2 hours and measure TR-FRET.
General Considerations

Assay Types
5.1.1 Endpoint Assay
The Transcreener® AMP2/GMP2 TR-FRET Red Assay is designed for endpoint readout.

5.1.2 Real-Time Assay
This assay can be performed in real time by eliminating stop reagents and including the AMP/GMP Detection Mixture components (antibody and tracer) in the enzyme reaction. However, this mode should only be used for relative activity comparisons, because the extended signal equilibration time precludes accurate quantitation of AMP/GMP. A standard curve run under similar conditions (continuous mode) will help in extrapolating the FRET ratios to amount of AMP or GMP product formed.
Reagent and Signal Stability
The Transcreener® technology provides a robust and stable assay method to detect AMP/GMP.

5.2.1 Signal Stability
The stability of the TR-FRET ratio assay window at 10% substrate conversion was determined after the addition of the AMP/GMP Detection Mixture to the standard samples. The ratio assay window at 10% substrate conversion (10 µM) remained constant (<10% change) for at least 24 hours at room temperature (20–25°C). If you plan to read TR-FRET on the following day, seal the plates to prevent evaporation.

5.2.2 AMP/GMP Detection Mixture Stability
It is important that the AMP/GMP Detection Mixture is prepared just prior to addition to the enzyme reaction. If you prepare the AMP/GMP Detection Mixture more than 30 minutes before addition, store it on ice or at 4°C until needed to help decrease the equilibration time.

5.2.3 Stopping the Reaction
We have inhibited the activity of several phosphodiesterases and ubiquitin ligases by the addition of Stop Buffer B (200 mM HEPES, 0.2% Brij®-35, and 400 mM EDTA [pH 7.5]), by quenching MgCl2 with EDTA. This buffer can be purchased separately: Part #2027 (1 mL) or 2032 (10 mL)
Troubleshooting

ProblemPossible Causes and Solutions
Low selectivitySuboptimal tracer concentration
  • To achieve maximum sensitivity and assay window, the AMP/GMP tracer concentration must be optimized for each starting ATP/cAMP/cGMP concentration.
ATP/cAMP/cGMP concentration out of range
  • Ensure that the starting ATP/cAMP/cGMP concentration is in the range of 1–1,000 µM.
No change in TR-FRET observedLow antibody/tracer activity
  • 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.
Interference from metal ions
  • Mn2+ or heavy metals like Cu2+, Fe2+, Fe3+, Cr3+, or Co2+ can quench terbium at higher concentrations. This effect can be relieved by using an EDTA-containing stop buffer (see Section 5 "Stopping the Reaction"). Use a minimum molar ratio of at least 4X EDTA to metal ions.
High background signal or change in signal after incubationNonspecific ATP/cAMP/cGMP hydrolysis
  • Aliquot the stock solution of nucleotide or prepare it fresh each time to avoid frequent freeze-thaw cycles.
Interference from impurities
  • Nuclease contamination in the buffer can cause the assay window to collapse, causing a change in FRET ratio. We recommend using nuclease-free water and freshly prepared buffer for each assay.
  • Some compounds may interfere with the detection mixture, causing a change in signal (see Section 7.2).
  • Bovine serum albumin (BSA) at concentrations >1% interferes with the detection reagents. Detergents, such as Brij-35, can be substituted for BSA in the enzyme reaction to prevent nonspecific binding of enzymes and substrates to the plate.

Appendix

Standard Curves
A standard curve (Figure 5) is required to convert FRET ratios to product formation (AMP or GMP) for quantitative data analysis. Because the Transcreener® AMP2/GMP2 TR-FRET Red Assay relies on a competitive binding reaction, the response is nonlinear, and therefore the signal is not directly proportional to reaction progress.
The wells for the standard curve should contain all AMP/GMP reaction components except the enzyme and receive AMP/GMP Detection Mixture. The curve is constructed to mimic an enzyme reaction: starting at the ATP/cAMP/cGMP concentration used for the screening reactions, ATP/cAMP/cGMP is decreased in increments and the AMP/GMP concentration is increased proportionally, keeping the sum of their concentrations [ATP/cAMP/cGMP + AMP/GMP] constant.
We recommend using a 12-point curve with concentrations of ATP/cAMP/cGMP and AMP/GMP corresponding to 0%, 0.5%, 1%, 2%, 3%, 5%, 7.5%, 10%, 15%, 30%, 50%, and 100% conversion (see Table 1). Allow 2-3 hours incubation prior to FRET measurement for complete equilibration.

% Conv.ATP (µM)AMP (µM)
1000100
505050
307030
158515
109010
7.592.57.5
5955
3973
2982
1991
0.599.50.5
01000
Table 1. Concentrations of ATP/AMP to prepare a 12-point standard curve.

Figure 5. Standard curves. Data are shown for ATP to AMP (A), cGMP to GMP (B), and cAMP to AMP (C) standard curves for initial substrate concentrations of 0.1–100 μM in the15 µL mock enzyme reaction. All assays were performed in 384-well plates (n = 12) and read on the Perkin Elmer EnVision multimode plate reader. ΔRatio is the change in TR-FRET 665:615 ratio from 0%.

Figure 5. Standard curves (cont.) D) Z’ values for initial velocity detection (10% conversion for 1 µM, 10 µM and 100 µM ATP/ADP standard curves, 30% for 0.1 µM) and lower limits of detection (LLD). LLD = the concentration of AMP/GMP that generates Z’ >0.

Use the following equations to calculate the Z' factor:



Summary of Additive Effects on the Transcreener® AMP2/GMP2 TR-FRET Assay
The assay window at 10% substrate conversion remains constant (<10% change) when up to 10% DMSO, DMF, ethanol, acetonitrile, ethanol, or methanol are used in the enzyme reaction. Contact BellBrook Labs Technical Service for further reagent compatibility information.


Not all combination of these components have been tested together. Results may vary depending on your assay conditions.

Bibliography
Antczak C, Shum D, Radu C, et al. Development and validation of a high-density fluorescence polarization based assay for the trypanosoma RNA triphosphatase TbCet1. Comb Chem High Throughput Screen 2009; 12(3): 258–268.
Huss KL, Blonigen PE, Campbell RM. Development of a Transcreener™ kinase assay for protein kinase A and demonstration of concordance of data with a filter-binding assay format. J Biomol Screen 2007;12(4): 578–584.
Kleman-Leyer KM, Klink TA, Kopp AL, et al. Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay. Assay Drug Dev Technol 2009;7(1): 56–65.
Klink TA, Kleman-Leyer KM, Kopp AL, et al. Evaluating PI3 kinase isoforms using Transcreener™ ADP assays. J Biomol Screen 2008;13(6): 476–485.
Liu Y, Zalameda L, Kim KW, et al. Discovery of acetyl-coenzyme A carboxylase 2 inhibitors: comparison of a fluorescence intensity-based phosphate assay and a fluorescence polarization-based ADP assay for high-throughput screening. Assay Drug Dev Technol 2007;5: 225–235.
Lowery RG, Kleman-Leyer KM. Transcreener™: screening enzymes involved in covalent regulation. Expert Opin Ther Targets 2006;10(1): 179–190.
Reifenberger JG, Pinghau G, Selvin PR. Progess in lanthanides as luminescent probes in Reviews in Fluorescence. Geddes CD, Lakowicz JR, eds. Vol. 2. 2005, Springer US, New York, pp 399–431.
Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 2009; 4(2): 67–73.
Contact Information
Email: [email protected] Phone: 608.443.2400 Toll-Free: 866.313.7881 FAX: 608.441.2967
Protocol references
Antczak C, Shum D, Radu C, et al. Development and validation of a high-density fluorescence polarization based assay for the trypanosoma RNA triphosphatase TbCet1. Comb Chem High Throughput Screen 2009; 12(3): 258–268.
Huss KL, Blonigen PE, Campbell RM. Development of a Transcreener™ kinase assay for protein kinase A and demonstration of concordance of data with a filter-binding assay format. J Biomol Screen 2007;12(4): 578–584.
Kleman-Leyer KM, Klink TA, Kopp AL, et al. Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay. Assay Drug Dev Technol 2009;7(1): 56–65.
Klink TA, Kleman-Leyer KM, Kopp AL, et al. Evaluating PI3 kinase isoforms using Transcreener™ ADP assays. J Biomol Screen 2008;13(6): 476–485.
Liu Y, Zalameda L, Kim KW, et al. Discovery of acetyl-coenzyme A carboxylase 2 inhibitors: comparison of a fluorescence intensity-based phosphate assay and a fluorescence polarization-based ADP assay for high-throughput screening. Assay Drug Dev Technol 2007;5: 225–235.
Lowery RG, Kleman-Leyer KM. Transcreener™: screening enzymes involved in covalent regulation. Expert Opin Ther Targets 2006;10(1): 179–190.
Reifenberger JG, Pinghau G, Selvin PR. Progess in lanthanides as luminescent probes in Reviews in Fluorescence. Geddes CD, Lakowicz JR, eds. Vol. 2. 2005, Springer US, New York, pp 399–431.
Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 2009; 4(2): 67–73.