Oct 29, 2025

Public workspaceTranscreener® GDP FI Assay Technical Manual

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6zp8dgqe/v1
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6zp8dgqe/v1
Protocol Citation: info 2025. Transcreener® GDP FI Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zp8dgqe/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 09, 2025
Last Modified: October 29, 2025
Protocol Integer ID: 229472
Keywords: assay development for new hts target, assay development, fluorescence reader, inhibitor profiling, throughput screening, inhibitor profiling across multiple target family, assay, therapeutic research laboratory, gdp detection, new hts target, selective for gdp, other substrate, used multimode plate reader, simple fluorescent intensity, multimode plate reader, complex multimode plate reader
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® GDP FI Assay extends the Transcreener® platform for GDP 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.
The assay is a red, competitive FI method (Figure 1). Because it is highly selective for GDP, the assay can be used with any enzyme that converts GTP to GDP, including small G proteins, regardless of what other substrates are used.
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® GDP FI Assay extends the Transcreener® platform for GDP 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.
The assay is a red, competitive FI method (Figure 1). Because it is highly selective for GDP, the assay can be used with any enzyme that converts GTP to GDP, including small G proteins, regardless of what other substrates are used.

Note
Caution: Many commercially available GTPase preparations contain GDP as a stabilizer, which must be removed or diluted prior to use with this assay so as not to contribute to background signal.
The Transcreener® GDP FI Assay provides the following benefits:
  • Accommodates GTP concentrations ranging from 0.1 µM to 1,000 µM.
  • Excellent data quality (Z’ ≥ 0.7) at low substrate conversion (typically ≤10%).
  • 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® GDP FI Assay. The Transcreener® GDP Detection Mixture contains a quenched ADP Alexa Fluor® 594 tracer bound to a GDP antibody conjugated to an IRDye® QC-1 quencher. GDP produced by the target enzyme displaces the tracer, which is no longer quenched and causes a positive increase in FI.

Product Specifications

ProductQuantityPart #
Transcreener® GDP FI Assay200 assays*3014-A
1,000 assays*3014-1K
10,000 assays*3014-10K
*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 (3014-A), or 384-well plates (3014-1K and 3014-10K) 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.
Materials Provided

ComponentCompositionNotes
GDP Antibody–IRDye® QC-11.4 mg/mL* solution in 100 mM KH2PO4(pH 8.5)The concentration of antibody needed for an enzyme target is dependent upon the GTP concentration and buffer conditions in the enzyme reaction (see Section 4.2). Sufficient antibody is included in the kit to complete 200 assays (Part # 3014-A), 1,000 assays (Part # 3014-1K), or 10,000 assays (Part # 3014-10K) at a GTP 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.

GDP Alexa Fluor® 594 Tracer800 nM solution in 2 mM HEPES (pH 7.5), 400 mM EDTA containing 0.01% Brij-35The final tracer concentration in the reaction is 4 nM
Stop & Detect Buffer B, 10X200 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 FI measurement is 0.5X.
GTP5 mMThe GTP supplied in this kit can be used for the enzyme reaction and to create a GDP/GTP standard curve, if desired.
GDP5 mMGDP is used to create the GDP/GTP 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
Caution: GTP is a common reagent in many laboratories; however, it is imperative that a highly purified preparation be used for the Transcreener® assay. If the GTP stock contains impurities, such as GDP, 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® GDP 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 FI of the Alexa Fluor® 594 tracer is required. The Transcreener GDP FI Assay has been successfully used on the following instruments: BMG Labtech PHERAStar Plus; Perkin Elmer EnVision®; Molecular Devices Spectramax M2; and Tecan and Safire2™.
  • 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® GDP 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® GDP 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 very 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 above in order to avoid spectral overlap.

Plate ReaderExcitation Filter/ Bandwidth Emission Filter/ BandwidthMirror ModuleOther Parameters
Envision (Perkin Elmer)545 nm/7 nm (Cat. # 2100-5070)635 nm/15 nm (Cat. # 2100-5590)D595Mirror: Texas Red FP single mirror Cat. # 2100-4190
PHERAstar Plus (BMG Labtech)580 nm/10 nm620 nm/10 nmNA
Safire2 (Tecan)580 nm/10 nm620 nm/10 nmNA Monochromator-based
SpectraMax M2 (Molecular Devices)584 nm612 nmNAEmission 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 GDP Alexa Fluor® 594 Tracer are excitation 590 nm (10 nm bandwidth) and emission 617 nm (10 nm bandwidth). The GDP 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.

The examples shown below are for an initial GTP concentration of 10 µM.

Low RFU Mixture Prepare the following solution:
ComponentStock ConcentrationFinal ConcentrationExample: 25 AssaysYour Numbers
GDP Antibody–IRDye® QC-11.4 mg/mL4.95 µg/mL1.8 µL
10X Stop & Detect Buffer B10X0.5X25.0 µL
GDP Alexa Fluor® 594 Tracer800 nM4 nM2.5 µL
Water470.7 µL
Total500.0 µL
The assay window will depend upon your initial GTP concentration. These volumes can be adjusted for fewer assays and different GTP concentrations.

High RFU Mixture
Prepare the following solution:
ComponentStock ConcentrationFinal ConcentrationExample: 25 AssaysYour Numbers
10X Stop & Detect Buffer B10X0.5X25.0 µL
GDP Alexa Fluor® 633 Tracer800 nM4 nM2.5 µL
Water472.5 µL
Total500.0 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.

Determine the Optimal GDP Antibody–IRDye® QC-1 Concentration
The Transcreener® GDP FI Assay requires detection of GDP in the presence of excess GTP (assuming initial velocity enzyme reaction conditions) using an antibody with a finite selectivity for the diphosphate vs. the triphosphate. The concentration of GDP Antibody–IRDye® QC-1 determines the total assay window and the GDP detection range; the amount needed primarily depends upon the GTP concentration in the enzyme reaction. To produce the most sensitive and robust assay signal, it is necessary to titrate the GDP Antibody–IRDye® QC-1 in the buffer system ideal for your enzyme or drug target.

Figure 3. Linear relationship between [GTP] and [GDP Antibody–IRDye® QC-1]. The antibody concentration can be calculated using the equation: y = 0.89x + 1.0.

4.2.1 Calculate the Antibody Concentration
As shown in Figure 3, the relationship between GTP and GDP Antibody–IRDye® QC-1 concentrations is linear. (Though shown for 0.1–100 µM GTP, the relationship is valid to 1,000 µM GTP.) Therefore, the quantity of GDP Antibody–IRDye® QC-1 for enzyme reactions that use between 0.1 μM and 1,000 μM GTP can be determined using the equation y = mx + b, where x = [GTP] (µM) in the enzyme reaction, y = [GTP Antibody–IRDye® QC-1] (µg/mL) in the 1X GDP Detection Mixture, m (slope) = 0.89, and b (y-intercept) = 1.0. We recommend a final reaction volume of 20 μL (384-well plate) or 50 µL (96-well plate).

For example, if you are using 3 µM GTP in a 10 µL enzyme reaction, the optimal GDP Antibody–IRDye® QC-1 concentration in the 1X GDP Detection Mixture (assuming 10 µL of GDP Detection Mixture was added to each 10 µL enzyme reaction) would be (0.89 × 3) + 1.0 = 3.67 µg/mL.

4.2.2 Optimize the Antibody Concentration
Using the GDP Antibody–IRDye® QC-1 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 titrating the antibody 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® GDP FI Assay. Use enzyme buffer conditions, substrate, and GTP 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 HEPES (pH 7.5), 4 mM MgCl2, 1% DMSO (test compound solvent), 0.01% Brij-35, and GTP. 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) × 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.
ComponentNotes
0% GTP Conversion ControlThis control consists of the GDP Detection Mixture, the enzyme reaction components (without enzyme), and 100% GTP (0% GDP). It defines the lower limit of the assay window.
100% GTP Conversion ControlThis control consists of the GDP Detection Mixture, the enzyme reaction components (without enzyme), and 100% GDP (0% GTP). It defines the upper 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., GTP) or acceptor substrate.
GDP/GTP Standard CurveAlthough optional, a GDP/GTP 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 ControlUse 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 GTP, 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 GDP Detection Mixture. 3. Prepare 1X GDP Detection Mixture as follows:

GTP Concentration: Examples
Component1 µM10 µM100 µMYour Numbers
GDP Antibody–IRDye® QC-113.5 µL70.7 µL642.9 µL
GDP Alexa Fluor® 594 Tracer100 µL100 µL100 µL
10X Stop & Detect Buffer B 1,000 µL 1,000 µL 1,000 µL
Water8,886.5 µL8,829.3 µL8,257.1 µL
Total10,000 µL10,000 µL10,000 µL
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). An example is shown below:
ABCD
GTP1 µM10 µM100 µM
GDP Antibody–IRDye® QC-11.89 µg/mL9.9 µg/mL90 µg/mL
4. Add 10 µL (384-well plates) or 25 µL (96-well plates) of 1X GDP Detection Mixture to the enzyme reaction. Mix using a plate shaker. 5. Incubate at room temperature (20–25°C) for 1 hour and measure FI.
4.4.2 GDP Detection Controls These controls are used to calibrate the FI plate reader and are added to wells that do not contain enzyme.

ComponentNotes
Minus Antibody (Free Tracer) ControlThis control contains the GDP Alexa Fluor® 594 Tracer without the GDP Antibody–IRDye® QC-1 and determines the maximum RFU value achievable.
Minus Tracer ControlThis control contains the GDP Antibody–IRDye® QC-1 without the GDP 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® GDP FI Assay is designed for endpoint readout. The Stop & Detect Buffer B contains EDTA to stop Mg2+-dependent enzyme reactions by chelating available Mg2+. The activity of some GTPase enzymes that produce GDP but do not require metal ions will not be stopped by the addition of the buffer provided.
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 GDP Antibody–IRDye® QC-1 is approximately 1 hour, making it difficult to quantitate GDP 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 GDP.
5.2.1 Signal Stability The stability of the FI assay window at 10% substrate conversion was determined after the addition of the GDP Detection Mixture to the standard samples. The RFU value at 10% substrate conversion (10 µM GTP) 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 GDP Detection Mixture Stability The GDP 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).
5.2.3 Solvent Compatibility The RFU window at 10% substrate conversion (10 µM GTP) remains constant (<10% change) when up to 10% DMSO, DMF, ethanol, acetonitrile, ethanol, or methanol are used in the enzyme reaction. Contact BellBrook Labs for further reagent compatibility information.
Troubleshooting

ProblemPossible Causes and Solutions
Low selectivitySuboptimal antibody concentration
  • Under the reaction conditions used in the Transcreener® GDP FI Assay, the GDP Antibody–IRDye® QC-1 is >140-fold selective for GDP over GTP. To achieve maximum sensitivity and assay window, the antibody concentration must be optimized for each starting GTP concentration.
GTP concentration out of range
  • Ensure that the starting GTP concentration is in the range of 1–1,000 µM.
No change in FI 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 signalNonproductive GTP hydrolysis
  • Certain enzymes catalyze some level of nonproductive GTP hydrolysis, to the extent that water is able to get into the active site. However, the rates are generally low even in the absence of acceptor substrate and are even further reduced when acceptor substrate is present. If you are using the assay to screen for potential acceptor substrates, then background from GTP hydrolysis has to be taken into account on a case-by-case basis. We recommend a “no substrate” control to detect nonproductive GTP hydrolysis.
Interference from impurities
  • Since the assay measures GDP production from any source, impurities that cause GDP production—such as a contaminating kinase, phosphatase, or GTPase—will interfere with accurate measurement of the desired GTPase activity. Care should be taken to minimize these potential contaminants in both GTPase and protein substrate preparations.

Appendix

Optimizing the GDP Antibody–IRDye® QC-1 Concentration
Using an antibody concentration calculated using the equation in Figure 3 (Section 4.2) will produce excellent results for most users. If it does not produce the results you require, we recommend that you titrate the GDP Antibody–IRDye® QC-1 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 antibody. We recommend using the EC10 concentration of antibody.

7.1.1 Titrate the GDP Antibody–IRDye® QC-1
1. Prepare the reaction buffer: 50 mM HEPES (pH 7.5), 4 mM MgCl2, and 0.01% Brij-35. Include GTP and substrate but omit the enzyme.
2. Dispense 10 µL (384-well plates) or 25 µL (96-well plates) of the reaction buffer into each well of columns 2–24.
3. Dispense 20 µL (384-well plates) or 50 µL (96-well plates) of GDP Antibody–IRDye® QC-1 (at the starting highest concentration in the same reaction buffer) into each well of column 1.
4. Remove 10 µL (384-well plates) or 25 µL (96-well plates) 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 (384-well plates) or 25 µL (96-well plates) of GDP Alexa Fluor® 594 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 FI.

7.1.2 Calculate the Optimal GDP Antibody–IRDye® QC-1 Concentration
The antibody concentration at the EC10 is often 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 fit into the equation below.

EC10 = (10 ÷ (100 – 10) )(1 ÷ hillslope) × EC50

Figure 5. GDP Antibody–IRDye® QC-1 titration at various GTP concentrations. The final 20 μL assay volume consisted of 4 nM GDP Alexa Fluor® 594 Tracer, 0.5X Stop & Detect Buffer B, 0.5X enzyme reaction mixture (50 mM HEPES [pH 7.5], 2 mM MgCl2, 0.5%  DMSO, 0.01% Brij-35, and GTP), and GDP Antibody–IRDye® QC-1 (n=3).

GDP/GTP Standard Curve
The standard curve mimics an enzyme reaction (as GTP concentration decreases, GDP concentration increases); the guanosine concentration remains constant. The GDP/GTP standard curve allows calculation of the concentration of GDP produced in the enzyme reaction and, therefore, the % GTP consumed (% GTP conversion). In this example, a 12-point standard curve was prepared using the concentrations of GDP and GTP shown in Table 2. Commonly, 8- to 12-point standard curves are used.

% Conv. GTP (µM)GDP (µM)
1000100
505050
307030
208020
158515
109010
7.592.57.5
5.095.05.0
2.597.52.5
1.099.01.0
0.599.50.5
01000
Table 2. Concentrations of GTP/GDP to prepare a 12-point standard curve.

Figure 6. GTP/GDP standard curves.

A) Sample data for 0.1 μM, 1 μM, 10 μM, and 100 μM GDP/GTP standard curves. The nucleotide concentration reflects the amount in the enzyme reaction, prior to the addition of the GDP 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 GDP/GTP standards) to 10 μL detection mix (GDP Antibody–IRDye® QC-1, 8 nM GDP Alexa Fluor® 594 Tracer, 1X Stop & Detect Buffer B) using the antibody concentration determined from Figure 3 (n = 24). The data are plotted as RFU vs. log [GDP] using 4-parameter nonlinear regression curve fitting.

B) Excellent Z’ values are obtained for the range of GTP concentrations used.

Use the following equation to calculate the Z’ factor:



Contact Information
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