Nov 06, 2025
Transcreener® UDP2 FI Assay Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Transcreener® UDP2 FI Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v95oyml3e/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 15, 2025
Last Modified: November 06, 2025
Protocol Integer ID: 229922
Keywords: transcreener udp2 fi assay, transcreener assay platform, transcreener udp2 antibody, assay development for new hts target, transcreener platform for udp detection, compatible with the assay, generic nature of the transcreener ht, assay development, fluorescence reader, transcreener ht, transcreener platform, universal biochemical ht, assay, inhibitor profiling, throughput screening, inhibitor profiling across multiple target family, fluorescence intensity, including glycosyltransferase, therapeutic research laboratory, udp detection, increase in fluorescence intensity, use of unmodified native substrate concentration, acetlygalactosyltransferase, galactosyltransferase, simple fluorescent intensity, new hts target, unmodified native substrate concentration, xylosyltransferase, udp
Abstract
The Transcreener assay platform 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 FI Assay extends the Transcreener platform for UDP detection by utilizing a simple fluorescent intensity (FI) output. It can be used on fluorescence readers typically found in academic and therapeutic research laboratories, as well as more complex multimode plate readers more commonly used in core facilities and HTS facilities. Texas Red filter sets are compatible with the assay.
The assay is a red, competitive FI method (Figure 1). The Transcreener UDP2 FI Assay is a universal biochemical HTS assay for enzymes that produce UDP, including glycosyltransferase, galactosyltransferase, glucuronyltransferase, N-acetylglucosamyltransferase, N-acetlygalactosyltransferase, xylosyltransferase, and glycogen, cellulose, lactose, and hyaluronan synthases. Enzyme activity is signaled by a increase in fluorescence intensity as the bound tracer is displaced from the Transcreener UDP2 Antibody-IRDye QC-1. The assay is a simple single step mix-and read format enabling the use of unmodified native substrate concentrations of 1 – 100 µM.
Troubleshooting
Introduction
The Transcreener assay platform 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 FI Assay extends the Transcreener platform for UDP detection by utilizing a simple fluorescent intensity (FI) output. It can be used on fluorescence readers typically found in academic and therapeutic research laboratories, as well as more complex multimode plate readers more commonly used in core facilities and HTS facilities. Texas Red filter sets are compatible with the assay.
The assay is a red, competitive FI method (Figure 1). The Transcreener UDP2 FI Assay is a universal biochemical HTS assay for enzymes that produce UDP, including glycosyltransferase, galactosyltransferase, glucuronyltransferase, N-acetylglucosamyltransferase, N-acetlygalactosyltransferase, xylosyltransferase, and glycogen, cellulose, lactose, and hyaluronan synthases. Enzyme activity is signaled by a increase in fluorescence intensity as the bound tracer is displaced from the Transcreener UDP2 Antibody-IRDye QC-1. The assay is a simple single step mix-and read format enabling the use of unmodified native substrate concentrations of 1 – 100 µM.
The Transcreener® UDP2 FI Assay provides the following benefits:
- Accommodates UDP concentrations ranging from 1 µM to 100 µM.
- Excellent data quality (Z’ ≥ 0.7) and signal at low substrate conversion using 1 µM UDP.
- 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.
- Red tracer further minimizes interference from fluorescent compounds and light scattering.
Figure 1. Schematic overview of the Transcreener UDP2 FI Assay. The Transcreener UDP Detection Mixture contains a quenched UDP Alexa Fluor 594 tracer bound to an UDP2 antibody conjugated to an IRDye QC-1 quencher. UDP produced by the target enzyme displaces the tracer, which is no longer quenched and causes a positive increase in FI.
Product Specifications
| Product | Quantity | Part # | |
| Transcreener® UDP2 FI Assay | 200 assays* | 3019-A | |
| 1,000 assays* | 3019-1K | ||
| 10,000 assays* | 3019-10K |
*The exact number of assays depends on enzyme reaction conditions. The kits are designed for use with 96-well plates using 50 µL reaction volumes (3019-A), or 384-well plates (3019-1K and 3019-10K) 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. Although not included in the kit, please be sure to aliquot and store UDP-sugar as directed by the manufacturer. UDP-sugars commonly breakdown and can result in poor assay quality. Be sure to avoid multiple freeze-thaw cycles.
Materials Provided
| Component | Composition | Notes | |
| UDP2 Antibody–IRDye® QC-1 | 1.4 mg/mL* solution in 100 mM KH2PO4 (pH 8.5) | The concentration of antibody needed for an enzyme target is dependent upon the UDP concentration and buffer conditions in the enzyme reaction (see Section 4.2). Sufficient antibody is included in the kit to complete 200 assays (Part # 3013-A), 1,000 assays (Part # 3013-1K), or 10,000 assays (Part # 3013-10K) at an UDP concentration up to 100 µM. Note: The Antibody IR-Dye QC-1 can have precipitate upon thawing. If so, spin down the precipitate and continue using the supernatant in the assay. The antibody will perform as directed. | |
| UDP Alexa Fluor® 594 Tracer | 800 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | The final tracer concentration in the reaction is 4 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 FI measurement is 0.5X. | |
| UDP | 5 mM | UDP is used to create a donor substrate/UDP standard curve. |
Note
*Note: The exact antibody concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration..
Note
Note: This kit contains sufficient reagents for up to 100 μM UDP. Please contact us if you plan on using greater than 100 μM UDP.
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® UDP2 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, enzyme cofactors, substrates, and test compounds.
- Plate Reader—A multi-detection microplate reader configured to measure FI of the Alexa Fluor® 594 tracer is required. The Transcreener UDP2 FI Assay has been successfully used on the following instruments: Perkin Elmer EnVision®; Molecular Devices Spectramax M2; and Tecan Infinite® M200 and Safire2™ (see Table 1).
- 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) and Corning 96-well, half-area plates (Cat. # 3686).
- 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 FI instruments.
Before You Begin
1. Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
2. Check the FI instrument and verify that it is compatible with the assay being performed (see Section 4.1).
Protocol
The Transcreener® UDP2 FI 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 final volume at the time that the plates are read; increase each volume to 25 µL (final volume 50 µL) if performing the assay in 96 well half-volume plates. 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. Volumes shown are for 384-well plates (and 96-well plates).
Set Up the Instrument
Becoming familiar with ideal instrument settings for FI is essential to the success of the Transcreener UDP2 FI Assay. Table 1 shows common instrument parameters.
Note that use of narrow bandwidth filters is critical for assay performance because the Stoke’s shift (separation between excitation and emission maxima) for the Alexa Fluor 594 is relatively narrow. It is possible to use wider bandwidth filters for some instruments, but it requires the use excitation and emission wavelengths different from those shown below in order to avoid spectral overlap.
| Plate Reader | Excitation Filter/ Bandwidth | Emission Filter/ Bandwidth | Mirror Module | Other Parameters | |
| Envision (Perkin Elmer) | 545 nm/7 nm (Cat. # 2100-5070) | 635 nm/15 nm (Cat. # 2100-5590) | D595 | Mirror: Texas Red FP single mirror Cat. # 2100-4190 | |
| PHERAstar Plus (BMG Labtech) | 580 nm/10 nm | 620 nm/10 nm | NA | ||
| Safire2 (Tecan) | 580 nm/10 nm | 620 nm/10 nm | NA | Monochromator-based | |
| SpectraMax M2 (Molecular Devices) | 584 nm | 612 nm | NA | Emission filter auto-cutoff at 610 nm |
Table 1. Instrument filters and settings for commonly used multimode plate readers. 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 FI
Ensure that the instrument is capable of measuring FI of Alexa Fluor 594. The optimal excitation/emission settings for the UDP Alexa Fluor 594 Tracer are excitation 590 nm (10 nm bandwidth) and emission 617 nm (10 nm bandwidth). The UDP Alexa Fluor 594 Tracer has been successfully used at excitations of 580–590 nm and emissions of 610–620 nm with bandwidths of 10 nm (see Table 1).
4.1.2 Define the Maximum FI Window for the Instrument
Measuring low (tracer + antibody) and high (free tracer) relative fluorescence units (RFUs) will define the maximum assay window of your specific instrument. Prepare Low and High RFU Mixtures in quantities sufficient to perform at least 6 replicates for each condition.
Use both tracer and antibody at 0.5X concentration in the final reaction volume. This mimics the 2-fold dilution when adding an equal volume of detection mixture to an enzyme reaction. As an example, if the calculated antibody concentration is 10 µg/mL, the concentration used here would be 5 µg/mL.
Low RFU Mixture
Prepare the following Low RFU Mixture as indicated in the table. Pipette 20 μL of the Total Low RFU Mixture to each well (from the example: 20 μL from 500 μL). Do not further dilute.
| Component | Stock Concentration | Final Concentration | Example: 25 Assays | Your Numbers | |
| UDP2 Antibody–IRDye QC-1 | 1.4 mg/mL | 5 µg/mL | 1.8 µL | ||
| 10X Stop & Detect Buffer B | 10X | 0.5X | 25.0 µL | ||
| UDP Alexa Fluor® 594 Tracer | 800 nM | 4 nM | 2.5 µL | ||
| Water | 470.7 µL | ||||
| Total | 500.0 µL |
The assay window will depend upon your initial UDP concentration. These volumes can be adjusted for fewer assays and different UDP concentrations.
High RFU Mixture
Prepare the following High RFU Mixture as indicated in the table. Pipette 20 μL of the Total High RFU Mixture to each well (from the example: 20 μL from 500 μL). Do not further dilute.
| Component | Stock Concentration | Final Concentration | Example: 25 Assays | Your Numbers | |
| 10X Stop & Detect Buffer B | 10X | 0.5X | 25.0 µL | ||
| UDP Alexa Fluor 594 Tracer | 800 nM | 4 nM | 2.5 µL | ||
| Water | 472.5 µL | ||||
| Total | 500.0 µL |
4.1.3 Measure the FI
Subtract the Low RFU Mixture readings from the corresponding High RFU Mixture readings. The difference between the low and high RFU values will give the maximum assay window. The values will differ, depending on the units from the plate reader, but the ratio (High RFU Mixture):(Low RFU Mixture) should be >5.0.
Note
Caution: Contact BellBrook Labs Technical Service for assistance if the ratio is <5.0.
Determine the Optimal UDP2 Antibody–IRDye QC-1 Concentration
The UDP2 Antibody-IRDye QC-1 is the only assay component that requires adjustment for different reaction conditions. Its concentration will define the dynamic range of the assay, and it should be adjusted based on the initial donor substrate concentration used in the enzyme reactions. To determined the optimal UDP2 Antibody-IRDye QC-1 concentrations up to 100 µM, perform an UDP2 Antibody-IRDye QC-1 titration using the reaction conditions for your enzyme or drug target.
4.2.1 Prepare a 500 μg/mL UDP2 Antibody Stock-IRDye QC-1 Solution
Make a stock solution for UDP2 antibody at 500 μg/mL in the enzyme reaction buffer.
4.2.2 Prepare the Enzyme Reaction Buffer
Prepare your enzyme reaction buffer without enzyme. The recipe for the enzyme reaction buffer depends upon the target enzyme.
4.2.3 Prepare Detection Mixture Containing UDP-Sugar Donor, Acceptor Substrate, UDP2 AlexaFluor 594 Tracer in Stop & Detect Buffer B.
Prepare 8 nM UDP2 AlexaFluor 594 Tracer in 1X Stop & Detect Buffer B with UDP-sugar and acceptor substrate (concentrations depend upon your target).
4.2.4 Titrate the UDP2 Antibody-IRDye QC-1 Stock Solution.
Dispense 20 μL of UDP2 Antibody-IRDye QC-1 Stock Solution into the column 1 wells. Dispense 10 μL of the enzyme reaction buffer across a 384-well plate (columns 2-24). Remove 10 μL from column 1 and serially titrate the contents across the plate (to column 24).
4.2.5 Add Detection Mixture
Dispense 10 μL of the Detection Mixture (containing UDP-sugar, acceptor substrate, 8 nM tracer, and 1X Stop & Detect Buffer B) to all wells on the assay plate from column 1 to column 24. Do not titrate.
4.2.6 Incubate and Measure Fluorescence Intensity
Mix the plate, equilibrate at room temperature (1 hour), and measure fluorescence intensity according to the instrument settings established in Section 4.1.
4.2.7 Plot RFU vs. log of UDP2 Antibody-IRDye QC-1 Concentration
The antibody concentration at the EC10 is used as a good compromise between sensitivity and maximal assay window. The EC10 is determined by inputting the EC50 and hillslope values from a sigmoidal dose response curve.
Figure 3. UDP2 Antibody-IRDye QC-1 titration at various UDP-GALNAc concentrations. Sample data for 1 μM, 10 μM, and 100 μM UDP-GALNAc binding curves. The nucleotide concentration reflects the amount in the enzyme reaction prior to the addition of the UDP Detection Mixture. 10 μL of Detection Mix was added to 10 μL the enzyme reaction mix.
4.2.8 Calculate Antibody Concentration for Detection Mixture
Use the following equation or results from your plot to calculate the EC10. The amount of antibody required in your 1X UDP Detection Mixture will be the EC10. Please note that the graphical example starts at an antibody concentration of 500 μg/mL in the 10 μL enzyme buffer. If graphing from the final concentration of 250 μg/mL in the 20 μL volume you will need 2X the EC10 in your 1X UDP Detection Mixture.
EC10 = (10 / (100-10))(1/hillslope) X EC50
From the example above: The EC50 was found to be 0.499 μg/mL. Using the equation (or graphing software) the EC10 is 2.956 μg/mL. Once 10 μL of UDP Detection Mix is added to the enzyme reaction the final concentration will be your original 0.5X EC10, or as in this example 1.478 μg/mL.
Optimize the Enzyme Concentration
Perform an enzyme titration to identify the optimal enzyme concentration for the Transcreener UDP2 FI Assay. Use enzyme buffer conditions, substrate, and UDP-sugar concentrations that are optimal for your target enzyme. If a compound screen is planned, you should include the library solvent at its final assay concentration. We routinely use enzyme buffer containing 50 mM TRIS (pH 7.5), 5 mM MgCl2, 1% DMSO (test compound solvent), and 0.01% Brij-35. Run your enzymatic reaction at its requisite temperature and time period. Refer to Section 5.2.3 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 FI 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) X EC50
Figure 4. Enzyme titration curve. The ideal range of enzyme concentrations is shown in red.
4.3.2 Enzyme Assay Controls
The enzyme reaction controls define the limits of the enzyme assay.
| Component | Notes | |
| 0% Conversion from UDP-Sugar Control | This control consists of the UDP Detection Mixture, the enzyme reaction components (without enzyme), and 100% UDP-Sugar (0% UDP). It defines the lower limit of the assay window. | |
| 100% Conversion to UDP Control | This control consists of the UDP Detection Mixture, the enzyme reaction components (without enzyme), and 100% UDP (0% UDP-Sugar). It defines the upper 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., UDP-Sugar) or acceptor substrate | |
| UDP/UDP-Sugar Standard Curve | Although optional, an UDP/UDP-Sugar 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 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 UDP-Sugar and acceptor substrate, then mix. The final volume of the enzyme reaction mixture should be 10 µL (384-well plates) or 25 µL (96-well plates). Incubate at a temperature and time ideal for the enzyme target before adding the ADP Detection Mixture.
3. Prepare 1X UDP Detection Mixture as follows:
| Component | Stock Concentration | Final Concentration | Example: 1000 Assays | Your Numbers | |
| UDP2 Antibody–IRDye QC-1 | 1.4 mg/mL | 2.956 µg/mL* | 21.1 µL | ||
| UDP Alexa Fluor® 594 Tracer | 800 nM | 8 nM | 100 µL | ||
| 10X Stop & Detect Buffer B | 10X | 1X | 1000.0 µL | ||
| Water | 8,878.9 µL | ||||
| Total | 10,000.0 µL |
*The EC10 obtained from Section 4.2. Your number will vary
Final concentrations in the detection mixture should be 8 nM tracer, 1X Stop & Detect Buffer B, and the antibody concentration calculated in Figure 3 (Section 4.2).
5. Add 10 µL (384-well plates) or 25 µL (96-well plates) of 1X UDP Detection Mixture to the enzyme reaction. Mix using a plate shaker.
6. Incubate at room temperature (20–25°C) for 1 hour and measure FI.
4.4.2 UDP Detection Controls
These controls are used to calibrate the FI plate reader and are added to wells that do not contain enzyme.
| Component | Notes | |
| Minus Antibody (Free Tracer) Control | This control contains the UDP Alexa Fluor® 594 Tracer without the UDP2 Antibody–IRDye® QC-1 and determines the maximum RFU value achievable. | |
| Minus Tracer Control | This control contains the UDP2 Antibody–IRDye® QC-1 without the UDP Alexa Fluor® 594 Tracer and is used as a sample blank. It contains the same antibody concentration in all wells. |
General Considerations
Assay Types
5.1.1 Endpoint Assay
The Transcreener® UDP2 FI 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 UDP2 Antibody–IRDye® QC-1 is greater than 15 minutes, making it difficult to quantitate UDP produced during short-term enzyme reactions. Note that the optimal antibody concentration may change when EDTA is omitted.
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 FI assay window at 10% substrate conversion was determined after the addition of the UDP Detection Mixture to the standard samples. The RFU value at 10% substrate conversion (10 µM UDP) remained constant (<10% change) for at least 24 hours at room temperature (20–25°C). If you plan to read FI on the following day, seal the plates to prevent evaporation.
5.2.2 UDP 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).
5.2.3 Solvent Compatibility
The RFU window at 10% substrate conversion (10 µM UDP) remains constant (<10% change) when up to 10% DMSO, DMF, ethanol, acetonitrile, ethanol, or methanol are used in the enzyme reaction. How solvents react to enzymes should be tested by the researcher. Contact BellBrook Labs for further reagent compatibility information.
Troubleshooting
| Problem | Possible Causes and Solutions | |
| Small Assay Window | Suboptimal antibody concentration
| |
| No change in FI observed | Low antibody/tracer activity
| |
| Does BSA Interfere with the assay? | Bovine Serum Albumin (BSA) interferes with the detection reagents and should be avoided. Detergent such as Brij-35 can be substituted in the enzyme reaction to prevent non-specific binding of enzymes and substrates to the plate, |
Appendix
UDP/UDP-Sugar Standard Curve
The standard curve mimics an enzyme reaction (as UDP-sugar concentration decreases, UDP concentration increases). The UDP/UDP-sugar standard curve allows calculation of the concentration of UDP produced in the enzyme reaction and, therefore, the % UDP-sugar consumed (% conversion). In this example, a 12-point standard curve was prepared using the concentrations of UDP-sugar and UDP shown in Table 2. Commonly, 8- to 12-point standard curves are used.
| % Conv. | UDP-sugar(µM) | UDP (µM) | |
| 100 | 0 | 100 | |
| 60 | 40 | 60 | |
| 40 | 60 | 40 | |
| 30 | 70 | 30 | |
| 20 | 80 | 20 | |
| 15 | 85 | 15 | |
| 10 | 90 | 10 | |
| 8.0 | 92 | 8.0 | |
| 6.0 | 94.0 | 6.0 | |
| 4.0 | 96.0 | 4.0 | |
| 2.0 | 98.0 | 2.0 | |
| 0 | 100 | 0 |
Table 2. Concentrations of UDP-sugar/UDP to prepare a 12-point standard curve.
Figure 6. UDP/UDP-sugar standard curves. A) Sample data for, 1 μM, 10 μM, and 100 μM UDP/UDP-sugar standard curves. The nucleotide % conversion concentration reflects the amount in the enzyme reaction, prior to the addition of the UDP Detection Mixture. Curves were obtained in a final 20 μL assay volume after adding 10 μL of reaction mix (50 mM HEPES [pH 7.5], 4 mM MgCl2, 1% DMSO, 0.01% Brij, and UDP-GALNAc/UDP standards) to 10 μL detection mix (UDP2 Antibody–IRDye® QC-1, 8 nM UDP Alexa Fluor® 594 Tracer, 1X Stop & Detect Buffer. The data are plotted using 4-parameter nonlinear regression curve fitting.
Z’ Values at Varying UDP-GALNAc Concentration
| % UDP Conversion | 1 µM UDP-GALNAc | 10 µM UDP-GALNAc | 100 µM UDP-GALNAc | |
| 0.5 | NA | 0.5 | 0.8 | |
| 1.0 | 0.0 | 0.6 | 0.8 | |
| 2.5 | 0.5 | 0.8 | 0.9 | |
| 5.0 | 0.7 | 0.9 | 0.9 | |
| 10 | 0.7 | 0.9 | 0.9 | |
| 100 | 0.8 | 0.9 | 0.9 |
Tabel 3. Z’ determination based on UDP-GALNAc Conversion using the antibody concentration determined from Figure 3 (n = 24). Excellent Z’ values are obtained for the range of UDP-GALNAc concentrations used.
Use the following equation to calculate the Z’ factor:
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