Oct 28, 2025
Transcreener® ADP² FP Assay Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Transcreener® ADP² FP Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9ok2gx1/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: September 26, 2025
Last Modified: October 28, 2025
Protocol Integer ID: 228302
Keywords: assay development for new hts target, assay development, selective for adp, inhibitor profiling, inhibitor profiling across multiple target family, throughput screening, assay, adp, enzyme, other substrate, new hts target, atpase, carbohydrate kinase, competitive fluorescence polarization, examples of enzyme
Abstract
The Transcreener® ADP2 FP Assay is a far-red, competitive fluorescence polarization (FP) assay (Figure 1). Because it is highly selective for ADP, the assay can be used with any enzyme that converts ATP to ADP, regardless of what other substrates are used. Examples of enzymes include protein, lipid, and carbohydrate kinases, ATPases, DNA helicases, carboxylases, and glutamine synthetase.
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.
Image Attribution
Figure 1. Schematic overview of the Transcreener® ADP² FP Assay. The Transcreener® ADP Detection Mixture contains an ADP Alexa Fluor® 633 tracer bound to an ADP² antibody. ADP produced by the target enzyme displaces the tracer, which rotates freely, causing a decrease in FP.
Materials
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® ADP² 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, buffer, acceptor substrate, MgCl2, or MnCl2, Brij-35, and test compounds.
- Plate Reader—A multidetection microplate reader configured to measure FP of the Alexa Fluor® 633 tracer is required. The Transcreener® ADP² FP Assay has 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 2–20 μL.
- Liquid Handling Devices—Use liquid handling devices that can accurately dispense a minimum volume of 2.5 μL into 384-well plates.
Troubleshooting
Safety warnings
Caution: ATP is a common reagent in many laboratories; however, it is imperative that a highly purified preparation be used for the Transcreener® assay. If the ATP stock contains impurities, such as ADP, the assay window will be compromised.
Note: Contact BellBrook Labs Technical Service for suppliers and catalog numbers for buffer components, and additional information regarding setup of FP instruments.
Caution: Contact BellBrook Labs Technical Service for assistance if the assay window is 3c150 mP.
Before start
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).
Introduction
The Transcreener® ADP2 FP Assay is a far-red, competitive fluorescence polarization (FP) assay (Figure 1). Because it is highly selective for ADP, the assay can be used with any enzyme that converts ATP to ADP, regardless of what other substrates are used. Examples of enzymes include protein, lipid, and carbohydrate kinases, ATPases, DNA helicases, carboxylases, and glutamine synthetase.
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® ADP2 FP Assay provides the following benefits:
- Accommodates ATP concentrations ranging from 0.1 µM to 1,000 µM.
- Excellent data quality (Z’ ≥ 0.7) and signal (≥85 mP polarization shift) at low substrate conversion (typically 10% or less) using 1 µM ATP.
- 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.
- Far-red tracer further minimizes interference from fluorescent compounds and light scattering.
Figure 1. Schematic overview of the Transcreener® ADP2 FP Assay. The Transcreener® ADP Detection Mixture contains an ADP Alexa Fluor® 633 tracer bound to an ADP2 antibody. ADP produced by the target enzyme displaces the tracer, which rotates freely, causing a decrease in FP.
Product Specifications
| Product | Quantity | Part # | |
| Transcreener® ADP2 FP Assay | 1,000 assays* | 3010-1K | |
| 10,000 assays* | 3010-10K |
*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.
Materials Provided
| Component | Composition | Notes | |
| ADP2 Antibody | 3.1 mg/mL solution in PBS with 10% glycerol* | The concentration of ADP2 Antibody needed for an enzyme target is dependent upon the ATP concentration and buffer conditions in the enzyme reaction (see Section 4.2). Sufficient antibody is included in the kit to complete 1,000 assays (Part # 3010-1K) or 10,000 assays (Part # 3010-10K) at an ATP concentration up to 100 µM. | |
| ADP Alexa Fluor® 633 Tracer | 400 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | The final tracer concentration in the 20 µL reaction is 2 nM. | |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35 | The Stop & Detect Buffer B components will stop enzyme reactions that require Mg2+. To ensure that the enzyme reaction is stopped completely, 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 at the time of FP measurement is 0.5X. | |
| ATP | 5 mM | The ATP supplied in this kit can be used for the enzyme reaction and to create an ADP/ATP standard curve, if desired. | |
| ADP | 5 mM | ADP is used to create the ADP/ATP standard curve. |
*The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.
Note
Caution: ATP is a common reagent in many laboratories; however, it is imperative that a highly purified preparation be used for the Transcreener® assay. If the ATP stock contains impurities, such as ADP, 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® ADP2 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, buffer, acceptor substrate, MgCl2 or MnCl2, Brij-35, and test compounds.
- Plate Reader—A multidetection microplate reader configured to measure FP of the Alexa Fluor® 633 tracer is required. The Transcreener ADP2 FP Assay has 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 2–20 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
- Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
- Check the FP instrument and verify that it is compatible with the assay being performed (see Section 4.1).
Protocol
The Transcreener® ADP2 FP Assay protocol consists of 4 steps (Figure 2). The protocol was developed for a 384-well format, using a 10 µL enzyme reaction and 20 µL complete assay 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 FP is essential to the success of the Transcreener® ADP2 FP Assay.
4.1.1 Verify That the Instrument Measures FP
Ensure that the instrument is capable of measuring FP (not simply fluorescence intensity) of Alexa Fluor® 633.
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 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 ADP Alexa Fluor® 633 Tracer and Stop & Detect Buffer B at 0.5X concentration 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 ADP Detection Mixture may contain 4 nM tracer. After adding this to the enzyme reaction, the concentration in the 20 µL complete assay would be 2 nM. Use 0.5X ADP2 antibody concentration calculated from Figure 3. The example here uses 10 µM ATP.
High FP Mixture
Prepare the following High FP Mixture as indicated in the table. Pipette 20 μL of the Total High FP Mixture to each well (from the example: 20 μL from 500 μL). Do not further dilute.
| Component | Stock Concentration | Complete Assay Concentration | Example: 25 Assays | Your Numbers | |
| ADP2 Antibody | 3.1 mg/mL | 5.9 µg/mL | 0.9 µL | ||
| 10X Stop & Detect Buffer B | 10X | 0.5X | 25.0 µL | ||
| ADP Alexa Fluor® 633 Tracer | 400 nM | 2 nM | 2.5 µL | ||
| Water | 471.6 µL | ||||
| Total | 500.0 µL |
The assay window will depend upon your initial ATP concentration. These volumes can be adjusted for fewer assays and different ATP concentrations.
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 Low FP Mixture as indicated in the table. Pipette 20 μL of the Total Low FP Mixture to each well (from the example: 20 μL from 500 μL). Do not further dilute.
| Component | Stock Concentration | Complete Assay Concentration | Example: 25 Assays | Your Numbers | |
| 10X Stop & Detect Buffer B | 10X | 0.5X | 25.0 µL | ||
| ADP Alexa Fluor® 633 Tracer | 400 nM | 2 nM | 2.5 µL | ||
| Water | 472.5 µL | ||||
| Total | 500.0 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 >150 mP.
Note
Caution: Contact BellBrook Labs Technical Service for assistance if the assay window is <150 mP.
Determine the Optimal ADP2 Antibody Concentration
The Transcreener® ADP2 FP Assay requires detection of ADP in the presence of excess ATP (assuming initial velocity enzyme reaction conditions) using an antibody with a finite selectivity for the diphosphate vs. the triphosphate. The concentration of ADP2 Antibody determines the total assay window and the ADP detection range; the amount needed primarily depends upon the ATP concentration in the enzyme reaction. To produce the most sensitive and robust assay signal, it is necessary to perform an ADP2 Antibody titration in the buffer system ideal for your enzyme or drug target.
Figure 3. Linear relationship between [ATP] and [ADP2 Antibody]. The antibody concentration can be calculated using the equation: y = 1.08x + 1.0
4.2.1 Calculate the Antibody Concentration
As shown in Figure 3, the relationship between ATP and ADP2 Antibody concentrations is linear. (Though shown for 0.1–100 µM ATP, the relationship is valid to 1,000 µM ATP.) Therefore, the quantity of ADP2 Antibody for enzyme reactions that use between 0.1 μM and 1,000 μM ATP can be determined using the equation y = mx + b, where x = [ATP] (µM) in the 10 μL enzyme reaction, y = [ADP2 Antibody] (µg/mL) in the 1X ADP Detection Mixture, m (slope) = 1.08, and b (y-intercept) = 1.0. We recommend a 20 µL complete assay for a 384-well plate (10 µL of the 1X ADP Detection mixture added to 10 µL of the enzyme reaction).
For example, if you are using 3 µM ATP in a 10 µL enzyme reaction, the optimal ADP2 Antibody concentration in the 10 µL 1X ADP Detection Mixture would be (1.08 × 3) + 1.0 = 4.24 µg/mL. In the 20 µL complete assay, the optimal ADP2 Antibody concentration would be half the concentration in the 1X ADP Detection Mixture, or 2.12 µg/mL for this example.
4.2.2 Optimize the Antibody Concentration
Using the ADP2 Antibody concentration calculated using the equation in Figure 3 will produce excellent results for most users. If it does not produce the results you require, refer to Section 7.1 for instructions on preparing an ADP2 Antibody titration in the buffer system ideal for your enzyme target.
Optimize the Enzyme Concentration
Perform an enzyme titration to identify the optimal enzyme concentration for the Transcreener® ADP2 FP Assay. Use enzyme buffer conditions, substrate, and ATP concentrations that are optimal for your target enzyme. If a compound screen is planned, you should include the library solvent at its complete assay concentration. We routinely use enzyme buffer containing 35 mM HEPES (pH 7.5), 4 mM MgCl2, 1% DMSO (test compound solvent), 0.015% Brij-35, and ATP. Run your enzymatic reaction at its requisite temperature and time period. Refer to Section 7.3 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 FP 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:
ECX = (X ÷ (100 – X) )(1 ÷ |hillslope| ) × EC50
Figure 4. 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.
4.3.2 Enzyme Assay Controls
The enzyme reaction controls define the limits of the enzyme assay.
| Component | Notes | |
| 0% ATP Conversion Control | This control consists of the ADP Detection Mixture, the enzyme reaction components (without enzyme), and 100% ATP (0% ADP). It defines the upper limit of the assay window. | |
| 100% ATP Conversion Control | This control consists of the ADP Detection Mixture, the enzyme reaction components (without enzyme), and 100% ADP (0% ATP). It defines the lower limit of the assay window. | |
| Minus-Nucleotide Control and Minus-Substrate Control | To verify that the enzyme does not interfere with the detection module, perform an enzyme titration in the absence of nucleotide (i.e., ATP) or acceptor substrate. | |
| ADP/ATP Standard Curve | Although optional, an ADP/ATP 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 7.2 for a description of how to run the standard curve. | |
| Background Control | Use only 0.5X enzyme reaction conditions and Stop & Detect Buffer B. |
Run an Assay
4.4.1 Experimental Samples
1. Add the enzyme reaction mixture to test compounds and mix on a plate shaker.
2. Start the reaction by adding ATP and acceptor substrate, then mix. The final volume of the enzyme reaction mixture should be 10 µL. Incubate at a temperature and time ideal for the enzyme target before adding the ADP Detection Mixture.
3. Prepare the 1X ADP Detection Mixture as follows:
ATP Concentration: Examples
| Component | 1 µM | 10 µM | 100 µM | Your Numbers | |
| ADP2 Antibody | 6.7 µL | 38.1 µL | 351.6 µL | ||
| ADP Alexa Fluor® 633 Tracer | 100 µL | 100 µL | 100 µL | ||
| 10X Stop & Detect Buffer B | 1,000 µL | 1,000 µL | 1,000 µL | ||
| Water | 8,893 µL | 8,862 µL | 8,548 µL | ||
| Total | 10,000 µL | 10,000 µL | 10,000 µL |
Final concentrations in the 1X ADP Detection mixture should be 4 nM tracer, 1X Stop & Detect Buffer B, and the ADP2 Antibody concentration calculated using the equation in Figure 3. An example is shown below:
y=1.08x+1.0
| A | B | C | D | |
| ATP | 1 µM | 10 µM | 100 µM | |
| ADP2 | 2.08 µg/mL | 11.8 µg/mL | 109 µg/mL |
4. Add 10 µL of 1X ADP Detection Mixture to 10 µL of the enzyme reaction. Mix using a plate shaker.
5. Incubate at room temperature (20–25°C) for 1 hour and measure FP.
4.4.2 ADP Detection Controls
These controls are used to calibrate the FP plate reader and are added to wells that do not contain enzyme.
| Component | Notes | |
| Minus Antibody (Free Tracer) Control | This control contains the ADP Alexa Fluor® 633 Tracer without the ADP2 Antibody and is set to 20 mP. | |
| Minus Tracer Control | This control contains the ADP2 Antibody without the ADP Alexa Fluor® 633 Tracer and is used as a sample blank for all wells. It contains the same ADP2 Antibody concentration in all wells. |
General Considerations
Assay Types
5.1.1 Endpoint Assay
The Transcreener® ADP2 FP Assay is designed for endpoint readout. The Stop & Detect Buffer B contains EDTA to stop Mg2+-dependent enzyme reactions by chelating available Mg2+.
5.1.2 Real-Time Assay
You can perform real-time experiments by substituting the Stop & Detect Buffer B, 10X (provided) with a detection buffer that does not contain EDTA. However, the equilibration time for the tracer and ADP2 Antibody is greater than 15 minutes, making it difficult to quantitate ADP produced during short-term enzyme reactions. Note that the optimal ADP2 Antibody concentration may change when EDTA is omitted.
Reagent and Signal Stability
The Transcreener® technology provides a robust and stable assay method to detect ADP.
5.2.1 Signal Stability
The stability of the FP assay window at 10% substrate conversion was determined after the addition of the ADP Detection Mixture to the standard samples. The mP value at 10% substrate conversion (10 µM ATP) remained constant (<10% change) for at least 24 hours at room temperature (20–25°C). If you plan to read FP on the following day, seal the plates to prevent evaporation.
5.2.2 ADP Detection Mixture Stability
The ADP Detection Mixture is stable for at least 8 hours at room temperature (20–25°C) before addition to the enzyme reaction (i.e., when stored on the liquid handling deck).
Troubleshooting
| Problem | Possible Causes and Solutions | |
| Low selectivity | Suboptimal antibody concentration
| |
| No change in FP observed | Low antibody/tracer activity
| |
| High background signal | Nonproductive ATP hydrolysis
|
Appendix
Optimizing the ADP2 Antibody Concentration
Using an antibody concentration calculated using the equation in Figure 4 will produce excellent results for most users. If it does not produce the results you require, we recommend that you perform an ADP2 Antibody titration in the buffer system ideal for your enzyme target. This titration will determine the optimal antibody concentration for your assay conditions. The nucleotide substrate concentration in the enzyme reaction generally determines the appropriate concentration of ADP2 Antibody. We recommend using the EC85 concentration of ADP2 antibody to add to the 1X ADP Detection Mixture.
7.1.1 Titrate the ADP2 Antibody
1. Prepare the reaction buffer: 35 mM HEPES (pH 7.5), 5 mM MgCl2, and 0.01% Brij-35. Include ATP and substrate but omit the enzyme
2. Dispense 10 µL of the reaction buffer into each well of columns 2–24.
3. Dispense 20 µL of ADP2 Antibody (at 2 mg/mL concentration in the same reaction buffer) into each well of column 1.
4. Remove 10 μL from each well of column 1 and add it to the corresponding well of column 2.
5. Repeat step 4 for the remaining columns, thereby performing a 2-fold serial dilution across the plate to column 24.
6. Add 10 µL of ADP Alexa Fluor® 633 Tracer (to a final concentration of 4 nM) in 1X Stop & Detect Buffer B to each well.
7. Mix the plate, equilibrate at room temperature for 1 hour, and measure FP.
7.1.2 Calculate the Optimal ADP2 Antibody Concentration
The antibody concentration at the EC85 is often used as a good compromise between sensitivity and maximal polarization value. The EC85 is determined by inputting the EC50 and hillslope values from a sigmoidal dose-response curve fit into the equation below. The ADP2 Antibody is added to the 1X ADP Detection Mixture at a concentration equivalent to 2 x [EC85].
EC85 = (85 ÷ (100 – 85) )(1 ÷ |hillslope|) × EC50
Figure 5. ADP2 Antibody titration at various ATP concentrations. The 20 µL complete assay consisted of 2 nM ADP Alexa Fluor® 633 Tracer, 0.5X Stop & Detect Buffer B, 0.5X enzyme reaction mixture (35 mM HEPES [pH 7.5], 2 mM MgCl2, 0.5% DMSO, 0.015% Brij-35, and ATP), and ADP2 Antibody (n=3).
ADP/ATP Standard Curve
The standard curve mimics an enzyme reaction (as ATP concentration decreases, ADP concentration increases); the adenine nucleotide concentration remains constant. The ADP/ATP standard curve allows calculation of the concentration of ADP produced in the enzyme reaction and, therefore, the % ATP consumed (% ATP conversion). In this example, a 12-point standard curve was prepared using the concentrations of ADP and ATP shown in Table 1. Commonly, 8- to 12-point standard curves are used.
| % Conv. | ATP (µM) | ADP (µM) | |
| 100 | 0 | 100 | |
| 50 | 50 | 50 | |
| 25 | 75 | 25 | |
| 15 | 85 | 15 | |
| 10 | 90 | 10 | |
| 7.5 | 92.5 | 7.5 | |
| 5.0 | 95.0 | 5.0 | |
| 3.0 | 97.0 | 3.0 | |
| 2.0 | 98.0 | 2.0 | |
| 1.0 | 99.0 | 1.0 | |
| 0.5 | 99.5 | 0.5 | |
| 0 | 100 | 0 |
Table 1. Concentrations of ATP/ADP to prepare a 12-point standard curve.
Figure 6. ATP/ADP standard curves.
A) Sample data for 0.1 μM, 1 μM, 10 μM, 100 μM, and 1,000 μM ADP/ATP standard curves. The nucleotide concentration reflects the amount in the enzyme reaction, prior to the addition of the 1X ADP Detection Mixture. Curves are obtained in a final 20 μL assay volume consisting of 35 mM HEPES (pH 7.5), 2 mM MgCl2, 1 mM EGTA, 0.5% DMSO, 0.015% Brij-35, 20 mM EDTA, 2 nM Alexa Fluor® 633 Tracer, ADP/ATP standards, and ADP2 Antibody (EC85 concentration) (n = 24). The data are plotted as mP vs. log [ADP] using 4-parameter nonlinear regression curve fitting. Alternatively, a 2-phase exponential decay and nonlinear regression can be used to present the data (GraphPad Prism).
B) Excellent Z’ values are obtained at <10% ATP conversion for the range of ATP concentrations used. Shown are 0.1 μM, 10 μM, and 1,000 μM ATP standard curves.
Use the following equations to calculate the Z’ factor:
Summary of Additive Effects on the Transcreener® ADP2 FP Assay
The assay window at 10% substrate conversion (10 µM ATP) 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.
a. <10% drop in ∆mP observed at the listed concentration and below.
b. mP at 0% or 10% increased or decreased <3 standard deviations of the plate controls at the listed concentration and below.
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 polarizationbased 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 highthroughput 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
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, Zalamena 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, Pinghua 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.