Nov 06, 2025

Transcreener® UDP2 FP Assay Technical Manual

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw42o9lmk/v1
  • 1BellBrook Labs LLC
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw42o9lmk/v1
Protocol Citation: info 2025. Transcreener® UDP2 FP Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw42o9lmk/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 10, 2025
Last Modified: November 06, 2025
Protocol  Integer ID: 229538
Keywords: assay development for new hts target, assay development, galactosyltransferase, glycosyltransferase, assay, inhibitor profiling, acetylgalactosyltransferase, enzyme, glucuronyltransferase, throughput screening, inhibitor profiling across multiple target family, examples of enzyme, acetylglucosamyltransferase, selective for udp, glycogen, competitive fluorescence polarization, other substrate, udp
Abstract
The Transcreener® UDP2 FP Assay is a far-red, competitive fluorescence polarization (FP) assay (Figure 1). Because it is highly selective for UDP, the assay can be used with any enzyme that produces UDP, regardless of what other substrates are used. Examples of enzymes include glycosyltransferases, galactosyltransferases, glucuronyltransferases, N-acetylglucosamyltransferases, N-acetylgalactosyltransferases, xylosyltransferases, and glycogen, cellulose, lactose, and hyaluronan synthases.
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.
Introduction
The Transcreener® UDP2 FP Assay is a far-red, competitive fluorescence polarization (FP) assay (Figure 1). Because it is highly selective for UDP, the assay can be used with any enzyme that produces UDP, regardless of what other substrates are used. Examples of enzymes include glycosyltransferases, galactosyltransferases, glucuronyltransferases, N-acetylglucosamyltransferases, N-acetylgalactosyltransferases, xylosyltransferases, and glycogen, cellulose, lactose, and hyaluronan synthases.
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® UDP2 FP Assay provides the following benefits:
  • Accommodates UDP concentrations ranging from 1 µM to 1,000 µM.
  • Excellent data quality (Z’ ≥ 0.7) at low substrate conversion (typically 10–30%).
  • 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® UDP2 FP Assay. The Transcreener® UDP Detection Mixture contains a UDP Alexa Fluor® 633 tracer bound to a UDP2 Antibody. UDP produced by the target enzyme displaces the tracer, which rotates freely, causing a decrease in FP.

Product Specifications

ProductQuantityPart #
Transcreener® UDP2 FP Assay1,000 assays*3018-1K
10,000 assays*3018-10K
*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

ComponentCompositionNotes
UDP2 Antibody3.1 mg/mL solution in PBS with 10% glycerol*The concentration of UDP2 Antibody needed for an enzyme target is dependent upon the UDP-sugar 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 # 3018-1K) or 10,000 assays (Part # 3018-10K) to detect UDP at a concentration up to 100 µM.
UDP2 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.
Stop & Detect Buffer B, 5X200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35The 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 0.25X.
UDP5 mMUDP is used to create the UDP-sugar/UDP standard curve.
*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® UDP 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 multi-detection microplate reader configured to measure FP of the Alexa Fluor® 633 tracer is required. The Transcreener UDP 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
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® UDP2 FP 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 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® UDP2 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 Alexa Fluor® 633.
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 UDP2 Alexa Fluor® 633 Tracer and Stop & Detect Buffer B at 0.25X concentration in a 20 µL complete assay. This mimics the 4-fold dilution when adding 5 µL of detection mixture to 15 µL of an enzyme reaction. As an example, the 1X UDP Detection Mixture may contain 16 nM tracer. After adding this to the enzyme reaction, the concentration in the 20 µL complete assay would be 4 nM.

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.

ComponentStock ConcentrationComplete Assay ConcentrationExample: 25 AssaysYour Numbers
UDP2 Antibody3.1 mg/mL5 µg/mL0.8 µL
5X Stop & Detect Buffer B5X0.25X25 µL
UDP2 Alexa Fluor® 633 Tracer800 nM4 nM2.5 µL
Water471.7 µL
Total500.0 µL
The assay window will depend upon your initial UDP-sugar concentration. These volumes can be adjusted for fewer assays and different UDP-sugar 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.

ComponentStock ConcentrationComplete Assay ConcentrationExample: 25 AssaysYour Numbers
5X Stop & Detect Buffer B5X0.25X25 µL
UDP2 Alexa Fluor® 633 Tracer800 nM4 nM2.5 µL
Water472.5 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 >150 mP. This measurement gives the maximal signal window that the instrument is capable of generating with these reagents. The assay window will be less than this value, and will depend on how far the UDP enzyme reaction proceeds.

Note
Caution: Contact BellBrook Labs Technical Service for assistance if the assay window is <150 mP.

Determine the Optimal UDP2 Antibody Concentration
The Transcreener® UDP2 FP Assay requires detection of UDP in the presence of excess UDP-sugar (assuming initial velocity enzyme reaction conditions) using an antibody with a finite selectivity for free UDP. The concentration of UDP2 Antibody determines the total assay window and the UDP detection range; the amount needed primarily depends upon the UDP-sugar concentration in the enzyme reaction. To produce the most sensitive and robust assay signal, it is necessary to perform a UDP2 Antibody titration in the buffer system ideal for your enzyme or donor sugar.
4.2.1 Titrate the UDP2 Antibody 1. Prepare the reaction buffer: 35 mM HEPES (pH 7.5), 5 mM MgCl2, and 0.01% Brij-35. Include the UDP-sugar and substrate but omit the enzyme. 2. Dispense 15 µL of the reaction buffer into each well of columns 2–24. 3. Dispense 30 µL of UDP2 Antibody (at 2 mg/mL in the same reaction buffer) into each well of column 1. 4. Remove 15 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 5 µL of UDP2 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.
4.2.2 Calculate the Optimal UDP2 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 UDP2 Antibody is added to the 1X UDP Detection Mixture at a concentration equivalent to 4 x [EC85].

EC85 = (85 ÷ (100 – 85) )(1 ÷ |hillslope|) × EC50

Figure 3. UDP2 Antibody titration at various UDP-glucuronic acid concentrations. The nucleotide concentration reflects the amount in the enzyme reaction prior to the addition of the 1X UDP Detection Mixture. The UDP2 Antibody (15 μL) in enzyme reaction mix (50 mM Tris [pH 7.5], 5 mM MgCl2, 8 mM EGTA, 1% DMSO, and UDP-glucuronic acid) was added to 5 μL of the 1X UDP Detection Mixture (16 nM UDP2 Alexa Fluor® 633 Tracer in 1X Stop & Detect Buffer B) (n = 2). The data are plotted as mP vs. log [UDP] using nonlinear regression curve fitting. The amount of antibody required in your UDP Detection Mixture for future experiments is 4 × EC85.

Optimize the Enzyme Concentration
Perform an enzyme titration to identify the optimal enzyme concentration for the Transcreener® UDP2 FP Assay. Use enzyme buffer conditions, substrate, and UDP-sugar concentrations that are optimal for your target enzyme and UDP2 Antibody 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 FP signal is ideal (EC50 to EC80) for screening of large compound libraries and generating inhibitor dose-response curves (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.

ComponentNotes
Negative (No Enzyme) ControlThis control is used to to calculate the decrease in FP caused by enzyme activity. It consists of 1X UDP Detection Mixture, the enzyme reaction components (without enzyme) and 100% UDP-sugar. It defines the upper limit of the assay window.
Positive (No Inhibitor) ControlThis control is used to determine the full activity of the enzyme being screened. It consists of UDP Detection Mixture, and the enzyme reaction components (including enzyme) but without any potential test compounds that might interfere with the full activity of the enzyme being screened. It defines the lower limit of the assay window.
Minus-Nucleotide ControlTo verify enzyme purity, perform an enzyme reaction in the absence of UDP-sugar.
UDP-Sugar/UDP Standard CurveAlthough optional, a UDP-sugar/UDP 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 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 Stop & Detect Buffer B.

Run an Assay
4.4.1 Experimental Samples 1. Prepare a master mix containing all UDP enzyme reaction components except the donor substrate and mix on a plate shaker. 2. Start the reaction by adding the donor substrate, 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 UDP Detection Mixture. 3. Prepare 1X UDP Detection Mixture as follows:

AStock ConcentrationDetection Mixture Concentration*ExampleYour Numbers
UDP2 Antibody3.1 mg/mL20 µg/µL*32.3 µL
UDP2 Alexa Fluor® 633 Tracer800 nM16 nM100 µL
5X Stop & Detect Buffer B5X1X1,000 µL
Water3,867.7 µL
Total5,000 µL
*Final concentrations in the 1X UDP Detection Mixture should be 16 nM tracer, 1X Stop & Detect Buffer B, and 4 × [EC85] UDP2 Antibody concentration as determined in Section 4.2.
4. Add 5 µL of 1X UDP 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 60 minutes and measure FP.

4.4.2 UDP 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 UDP2 Alexa Fluor® 633 Tracer without the UDP2 Antibody and is set to 20 mP.
Minus Tracer ControlThis control contains the UDP2 Antibody without the ADP Alexa Fluor® 633 Tracer and is used as a sample blank for all wells. It contains the same UDP2 Antibody concentration in all wells.

General Considerations

Assay Types
5.1.1 Endpoint Assay The Transcreener® UDP2 FP Assay is designed for endpoint readout. The Stop & Detect Buffer C contains EDTA to stop Mg2+-dependent enzyme reactions by chelating available Mg2+. Contact BellBrook Labs regarding stop buffers for non-Mg2+-dependent enzymes.
5.1.2 Real-Time Assay You can perform real-time experiments by adding the UDP Detection Mixture, without the Stop & Detect Buffer B, directly to an enzyme reaction at initiation of the reaction. UDP detection equilibration time is not instantaneous, making it difficult to quantify UDP production; however, this method can provide insight into optimal enzyme concentration and incubation time. Note that the optimal UDP2 Antibody concentration may change when EDTA is omitted from the reaction.
Reagent and Signal Stability
The Transcreener® technology provides a robust and stable assay method to detect UDP.
5.2.1 Signal Stability The stability of the FP assay window at 10% substrate conversion was determined after the addition of the UDP Detection Mixture to the standard samples. The mP value at 10% substrate conversion (10 µM UDP-glucuronic acid) 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 UDP Detection Mixture Stability The UDP Detection Mixture is stable for at least 24 hours at room temperature (20–25°C) before addition to the enzyme reaction (i.e., when stored on the liquid handling deck).
Troubleshooting

ProblemPossible Causes and Solutions
Low selectivitySuboptimal antibody concentration
  • To achieve maximum sensitivity and assay window, the UDP2 Antibody concentration must be optimized for each starting UDP-sugar concentration.
UDP-sugar concentration out of range
  • Ensure that the starting UDP-sugar concentration is in the range of 1–100 µM.
No change in FP observedLow antibody/tracer activity
  • The tracer and antibody are stable for up to 10 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.
High background signalInterference from impurities
  • Since the assay measures UDP-sugar conversion from any source, impurities that cause UDP production—such as a contaminating enzyme—will interfere with accurate measurement of the desired enzyme activity. Care should be taken to minimize these potential contaminants in both UDP-sugar and substrate preparations.

Appendix

UDP-Sugar/UDP Standard Curve
The standard curve mimics an enzyme reaction (as UDP-sugar concentration decreases, UDP concentration increases). The UDP-sugar/UDP standard curve allows calculation of the concentration of UDP produced in the enzyme reaction and, therefore, the % UDP-sugar consumed (% UDP-sugar conversion). In this example, a 12-point standard curve was prepared using the concentrations of UDP-glucuronic acid and UDP shown in Table 1. Commonly, 8- to 12-point standard curves are used.

% Conv.UDP-Glucuronic Acid (µM) UDP (µM)
1000100
505050
307030
158515
109010
7.592.57.5
5955
3973
2982
1991
0.599.50.5
01000

Figure 5. UDP-glucuronic acid/UDP standard curves. A) Sample data was plotted for for 1 μM, 10 µM, and 100 μM UDP-glucuronic acid/UDP standard curves. The nucleotide concentration reflects the amount in the enzyme reaction, prior to the addition of the 1X UDP Detection Mixture. A polarization shift of 60–100 mP and a Z’ value of 0.5 indicates robust assay performance for HTS applications. For the initial UDP-glucuronic acid concentration of 1 µM, these criteria were achieved at <7.5% conversion; for 10 µM UDP-glucuronic acid, at 2% conversion ; and for 100 µM UDP-glucuronic acid concentration, at 0.75% conversion.

Use the following equations to calculate the Z’ factor:



Summary of Additive Effects on the Transcreener® UDP2 FP Assay
The assay window at 10% substrate conversion (10 µM UDP-sugar) 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.

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Contact Information
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