TR-FRET utilizes time-gated fluorescence intensity measurements to quantitate molecular association or dissociation events. FRET is the non-radiative energy transfer between a fluorophore and a chromophore. This transfer of energy is distance-dependent and requires an overlap of the donor emission and acceptor absorption spectra. Time resolved fluorescence (TRF) measurements allow distinction between short- and long-lived fluorescent signals following excitation by a short pulse of light. The immediate “prompt” fluorescence dissipates in the first few nanoseconds allowing a “delayed” measurement under conditions of nearly zero background. Consequently, the combination of FRET based assays and TRF measurements allow very sensitive detection of binding/dissociation events in a homogeneous format.
Common Applications Using TR-FRET
TR-FRET can be applied to assays to detect the association of two molecules, as in receptor-ligand or protein-protein interactions, or when they are separated, as in the two ends of a protease substrate after cleavage. The most commonly used labels are the long-lived lanthanide europium (donor) and the short-lived acceptor protein allophycocyanin (APC also
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 2
known as XL665). Lanthanide metals are quenched in aqueous environments and thus require protection in the form of a proprietary chelate or cryptate. Commercially available reagents for TR-FRET can be obtained from Packard (employs the cryptate, tradename HTRF) and Perkin Elmer (fomerly known as EG&G Wallac, employs the chelate, tradename LANCE).
Guidelines forDeveloping a TR-
FRET Assay
This application note presents data for the development of a model TR-FRET assay using europium cryptate labeled biotin (Biot-K) and allophycocyanin labeled streptavidin (SA-XL665). Additional data will be presented on the detection of tyrosine kinase (TK) enzyme products. The data support the following guidelines for development of a TR-FRET assay.• Define assay performance specifications.• Determine concentration of donor required to meet performance
specifications.• Optimize donor/acceptor concentrations to achieve acceptable assay
sensitivity and dynamic range.• Verify assay performance/specificity.• Optimize other parameters: an iterative process to reevaluate enzyme
assay conditions, followed by re-optimization of detection reagents.
Equipment and Materials
RecommendedEquipment and
Materials
The following materials were used in this application note for performing the TR-FRET assay.
Table 1. Equipment and materials:
Item Source
Criterion™ System with Release 2.0 Soft-ware—one of the following:
• Analyst™ AD or HT• Acquest™• ScreenStation™
Please contact your local Molecular Devices Corpora-tion representative for the instrument best suited to meet your needs.
a. Multi-method allows you to specify two previously defined TRF
methods. This enables you to specify a single read command that
yields values for both donor and acceptor wavelengths. In addition,
you can select automatic ratio calculations of these values.
Excitation and emission filters See above
Dichroic mirror Dichroic UV, see Table 2 above
Z-height 3 mm (set)
Attenuator Out
Delay after flash 200 µsec donor, 50 µsec acceptor
Integration time 1000 µsec donor, 150 µsec acceptor
Lamp Flash, 1000V
Flashes per well 100
Time between flashes 10 msec
Raw data units Counts
Plate settling time 10 msec
PMT setup digital
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 4
Defining AssayPerformance
Specifications
Prior to assay development, defining the assay requirements will enable a clear-cut decision as to the feasibility of the developed assay for the high-throughput environment.
The following are some parameters to consider when developing an assay for high-throughput screening:
Assay Robustness
Robustness is the ability to withstand interfering effects, such as temperature, time, and volume variations. Some quantitative measurements for assay robustness are:• Lower limit of detection• Number of assay steps• Dynamic range• Assay precision
Note
Many of these quantitative criteria are assessed in the calculation of an assay value ratio (AVR) or a related measurement of assay performance called a z-factor
(Zhang
et al.
and Li
et al.
)
.
Assay Scalability
Cost considerations become magnified as screening libraries become larger. For this reason, assay scale ability may be an important factor in assay development.
Assay Time
As the number of targets to screen become larger, the time to first or subsequent result may be a critical factor.
Assay Development Recommendations
Overview
To develop a TR-FRET assay, we performed a series of experiments that optimized the essential elements in successive steps. These experiments addressed the following assay requirements:• Donor concentration required to meet performance specifications• Donor/acceptor concentration required to achieve acceptable assay sen-
sitivity and dynamic range• Assay performance and specificity• Further optimization strategies
Determining theRequired
Concentration ofDonor
The following describes a procedure and presents the results of an experiment that was used to determine the concentration of donor required for an assay.
Procedure for determining the required concentration of donor:
Step Action
1
Prepare a dilution series of Eu labeled biotin (donor) under the conditions that will approximate those of the actual assay.Include buffer only wells (all components except the Eu-labeled biotin) to evaluate the background signals.
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 5
Table 5 summarizes the data collected. The criteria used for selection of a concentration are the following:• 620 nm signal between 10,000–100,000 counts• CV of <10%• Donor spillover in the 665 acceptor emission channel <10%
Note
Due to cost considerations, continued assay development will attempt to minimize the concentration of Eu-labeled donor required.
Results are in counts/100 flashes with CV’s in parenthesis (n=5). Signal exceeding 1000
counts/flash are saturating the digital circuitry.
From the summary results in Table 4, it is apparent that 5 nM Biot-K meets the selection criteria. The next step in the optimization process will also include 10 nM Biot-K for comparison.
Optimizing theDonor/AcceptorConcentrations
Optimization of the donor/acceptor concentration will help to achieve acceptable assay sensitivity and dynamic range.
To optimize the acceptor concentration for detection of the ligand (biotin), a matrix of acceptor and donor concentrations were run in the presence of excess unlabeled ligand (representing minimal or background FRET) and no ligand (representing maximal FRET). In addition to the matrix of donor and acceptor concentrations to be evaluated, background controls of the detection reagents should be characterized at the same time.
Energy transfer for each condition was calculated as the ratio of acceptor to donor signals and multiplied by a factor of 10,000 for ease of data manipulation. Alternatively, the acceptor signal at 665 nm can be used to monitor the efficiency of energy transfer.
Assay dynamic range was calculated as the ratio of ratios. The calculated acceptor to donor ratio under conditions of maximal energy transfer will be divided by the calculated acceptor to donor ratio under conditions of minimal energy transfer.
2
Read the plate at the recommended instrument settings for both the donor and acceptor emission wavelengths.Run at least four replicates per data point to obtain an approxi-mation of the imprecision associated with each signal level.
Table 4. Summary of results:
Sample 620 nm 665 nm
TR-FRET buffer 95 (8.4%) 52 (21%)
1.25 nM Biot-K 8,740 (16%) 85 (13%)
5 nM Biot-K 39,118 (4.5%) 219 (9.7%)
20 nM Biot-K 117,021 (4.8%) 634 (8.8%)
80 nM Biot-K 432,445 (13%) 3635 (28%)
Procedure for determining the required concentration of donor: (Continued)
Step Action
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 6
Results are in counts/100 flashes. Signal exceeding 1000 counts/flash are saturating the
digital circuitry. Each value is an average of three replicates.
Evaluating theData
Controls
Control wells can be identified in Table 5 by the lack of a calculated ratio (reported as n/r or not relevant). As expected, when no donor is present, we see very low 620 nm signals. The 665 nm background increases with increasing acceptor concentrations without the presence of a donor. This increase accounts for <10% of the 665 nm signal representing maximal FRET conditions. The ‘spillover’ of donor signal into the acceptor channel is minimal as seen by the low acceptor signal (665 nm) levels when the acceptor molecule (SA-XL665) is not present.
The donor only control (Biotin-K) signal at 620 nm serves as a reference point for comparison to 620 nm assay signals where energy transfer is present. When energy transfer is present, a decrease in the 620 nm donor emission is expected since donor photon emission is decreased by the non radiative energy transfer to the acceptor. The acceptor only control signal at 665 nm should be minimal or close to the buffer background since its fluorescence lifetime is on the order of nanoseconds and all the assays signals are measured using time-gated fluorescence intensity. However, when energy transfer is present, an increase in the 665 nm acceptor emission is expected. The non-radiative energy transfer from the long-lived lanthanide
Table 5. Results for optimization of donor and acceptor concentrations:
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 7
donor (~1 ms fluorescent lifetime) to the short-lived acceptor protein APC (SA-XL665) results in an apparent increase of the acceptor lifetime allowing time-gated fluorescent intensity measurements of acceptor signal.
Evaluating the Raw Data
The data demonstrate that there is very efficient energy transfer between the donor (Biotin-K) and acceptor (SA-XL665) because of:• A marked decrease in donor signal (620 nm) with increasing concentra-
tion of acceptor.• A marked increase in acceptor signal (665 nm) with increasing concen-
trations of acceptor.• A plateau at equimolar concentrations of acceptor and donor.
The data demonstrates specificity because of:• A marked inhibition of energy transfer with the addition of unlabeled
ligand (Biotin).
Note
The increase in background signal in the presence of excess unlabeled biotin is probably the result of diffusion enhanced FRET and not biological binding events. Diffusion enhanced FRET is apparent when the donor has a long lifetime, allowing an extended period of time in which the acceptor has the ability to randomly diffuse within the required distance for energy trans-fer.
Evaluating the Dynamic Range
Figure 1 summarizes two different approaches to evaluating assay dynamic range:• Ratio• Delta
Ratio Method
The ratio method uses the acceptor/donor ratio under conditions of maximal FRET and divides that by the acceptor/donor ratio under conditions of minimal or background FRET. If the dynamic range is calculated from the ratio of ratios, the background FRET associated starts to have a negative impact on the assay dynamic range with increased acceptor concentrations.
Note
CIS bio international holds patents covering the ratio method using lanthanide chelates and fluorescent energy transfer. It is the understanding of Molecular Devices Corporation that users purchasing reagents from Packard Biosciences, a licensee of the CIS bio patents, are free to use any instrument with these reagents. For further questions concerning the scope and cover-age of this patent, users should seek legal opinion.
Delta Method
The delta method uses the difference of ratios. The minimal FRET ratio is subtracted from the maximal FRET ratio. If the dynamic range is evaluated using the difference of ratio’s (delta), there is a steep increase in dynamic range until approximately equal molar ratios of donor and acceptor are present, and it plateaus at higher acceptor concentrations.
Conclusions
When calculated by the ratio method, the background FRET associated with the higher acceptor concentrations begins to have a negative impact on the dynamic range.
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 8
When calculated by the delta method, the dynamic range plateaus at the higher acceptor concentrations.
Figure 1. Two approaches to evaluating assay dynamic range
The assay was performed in an HE 384-well white microplate with a final assay volume of 12 µL/well. The assay was performed as follows: addition of 4 µL/well of 3X the appropriate acceptor concentration (SA-XL665), addition of 4 µL/well of 3X the appropriate concentration of d-biotin (see Table 5 on page 6), 15 minutes of incubation at room temperature, addition of 4 µL/well of 15 nM donor (final concentration 5 nM biotin-K), 15 minutes of incubation at room temperature, and reading the assay plate at both the donor and acceptor wavelengths
using the recommended instrument settings.
Verifying AssayPerformance and
Specificity
Calibration Curve
Use the donor/acceptor optimization data to prepare a calibration curve. Five nM donor was selected because it had comparable results to 10 nM at a lower cost. Biotin calibration curves were prepared at three different acceptor concentrations to show the impact on assay sensitivity. The results, shown in Figure 2, indicate that the concentration of d-biotin necessary to modulate the ratio is a function of the acceptor (SA-XL665) concentration.
Figure 2. Calibration curve for TR-FRET model assay
The assay was performed in an HE 384-well white microplate with a final volume of 12 µL/well. Each data point represents the average of five replicate wells. The assay was performed as follows: addition of 4 µL/well of 3X the appropriate acceptor concentration (SA-XL665), addition of 4 µL/well of 3X the appropriate concentration of d-biotin, 15 minutes of incubation at room temperature, addition of 4µL/well of 15 nM donor (final concentration of 5 nM biotin-
ratio
delta
20
30
40
50
60
70
80 12
10
8
6
4
21 2 4 8 16 32 64 128 256
Rat
io
Del
ta (x
103 )
SA-XL665 (nM)
10
2
4
6
8
10
12
14
16
1 10 100 1000 10,000
8 nM
32 nM
128 nM
d-biotin (nM)
TR-F
RET
ratio
(cou
nts/
coun
ts x
103 )
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 9
K), 15 minutes incubation at room temperature, and read the assay plate at both donor and
acceptor wavelengths using the recommended instrument settings.
FurtherOptimization
Strategies
Working with a model TR-FRET system such as the one presented here is often beneficial because it can:• Enable you to become familiar with TR-FRET chemistry and instru-
ment parameters.• Allow you to optimize many parameters that may be transferable to
another TR-FRET system.
Note
Keep in mind when comparing assays types that the apparent life-times of the bound donor and acceptor are fixed and depend on the structure of the complex.
Alterations in instrument settings and assay conditions may result in an improvement of the following:• Dynamic range• Decrease in spillover and background• Better coefficients of variation• Increased magnitude of desired signals
Some of the instrument settings and assay conditions that can be evaluated further are the following:• Evaluation of filters—For example, wider filters can be used to increase
the magnitude of raw signals.• Variation of z-heights—The volume used will dictate the optimal z-
height for the most sensitive measurement.• Evaluation of the relationship between the number of flashes and preci-
sion—For faster reads requiring fewer flashes, an increase in concentra-tion of reagents may be essential to maintain a required level of precision.
• Evaluation of plate manufacturers—Different plates have reduced back-ground and binding of reagents.
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 10
Tyrosine Kinase Application
Overview
The following section presents the results of a TR-FRET screening assay that was developed applying the techniques described in the model assay system. The assay is designed to detect a tyrosine kinase enzyme product. A schematic of the assay is shown in Figure 3.
Figure 3. The principles of tyrosine kinase TR-FRET assay
Defining AssaySpecifications
Before optimizing the acceptor concentration, the assay conditions were defined:
• 4 µL total volume for enzyme portion of the assay• 2 hours incubation at room temperature• Reaction stopped with EDTA in the TR-FRET buffer with the detection
reagents PY20-K and SA-XL665• Final assay volume is 20 µL• Consideration of DMSO tolerance and the impact of temperature fluc-
tuation
The requirement of the 1 µM biotinylated substrate for the enzyme portion of the assay puts constraints on the minimum concentration of the ‘generic’ SA-XL665 (acceptor) that must be present in the detection portion of the assay. Theoretically, there are 4 binding sites for biotin on each molecule of
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 11
streptavidin. Thus, for energy transfer to take place, the Eu-labeled anti-phosphotyrosine antibody (donor) must bind to a complex of biotinylated, phosphorylated substrate and acceptor. If insufficient concentration of acceptor is present, an incomplete complex of donor and biotinylated, phosphorylated substrate can be formed and assay signal is lost (energy transfer is not present). In addition to the constraint of a minimum concentration for the “generic” acceptor required, care should be taken to avoid an excess of acceptor that can contribute to non-specific, background energy transfer resulting in a decreased assay dynamic range.
Optimizing theDonor/AcceptorConcentrations
A titration of PY20-K (donor) was evaluated under conditions representative of final assay configuration. Acceptable donor signal level (> 10,000 counts in 100 flashes but < 100,000) was achieved at a concentration of 3 nM. Next, the acceptor concentration was optimized for detection of enzyme product. A matrix of acceptor and donor concentrations were run in the presence of maximal enzyme product and no enzyme product. Maximal enzyme product was obtained under the enzyme assay conditions listed above, yielding maximal energy transfer upon addition of the TR-FRET detection reagents. Minimal enzyme product will be obtained by omitting the ATP from the enzyme reaction, yielding minimal or background energy transfer upon addition of the TR-FRET detection reagents.
Verifying AssayPerformance and
Specificity
Following the optimization of the detection portion of the assay, the enzyme portion of the assay was run in the presence of various concentrations of staurosporine to determine an IC
50
value for comparison to literature values. Figure 4 summarizes these results. The calculated IC50 value of ~700 nM agrees favorably with literature values.
Figure 4. Tyrosine kinase screening assay
The assay was performed in an HE 384-well white microplate with a final assay volume of 20 µl/well. The enzyme portion of the assay was prepared, in bulk, in a v-bottom polypropylene plate. The enzyme reaction buffer consisted of 50 mM TRIS, 2 mM MnCl
2
, 10 mM MgCl
2
, 0.1% BSA at a pH 7.6, with enzyme at 2 nM, ATP at 50 µM, biotinylated-peptide substrate at 1 µM and staurosporine at various concentrations. From the bulk preparation of enzyme reactions, 4 samples of 4 µl each were transferred from each polypropylene well into the wells of an HE 384-well white microplate, yielding 16 replicate samples for each condition. Following a 2 hour room temperature incubation, the detection reagents were added yielding a final assay volume of 20 µl/well. The final concentration of detection reagents was 3 nM PY20-K and 12.5 nM SA-XL665. The detection buffer consisted of 50 mM TRIS, 25 mM EDTA, 500 mM KF, 0.2% BSA at a pH7.6 yielding a final concentrations of 20 mM EDTA and 400 mM KF. After the addition of the detection reagents, there was a 30 minute room tem-
150
200
250
0.01 0.1 1.0 10Staurosporine (µM)
TR-F
RET
ratio
(cou
nts/
coun
ts)
Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 12
perature incubation followed by reading the assay plate at both donor and acceptor wave-
lengths as defined in the recommended instrument settings section.
FurtherOptimization
The assay could be further enhanced by optimizing the following:• Substrate used• ATP concentration• Enzyme concentration
Note
For this assay, two other polymer substrates were tested: biotin-polyGT and biotin-polyGAT. Neither of them performed as well as the peptide substrate.
The development of the tyrosine kinase screening assay presented some issues not present in the model system:• The use of the generic detection reagent SA-XL665 resulted in less effi-
cient energy transfer than what was observed with the model assay.• The minimal requirement for SA-XL-665 capture of the potentially
phosphorylated substrate resulted in an increase in the background FRET levels compromising the assay dynamic range.
Some strategies that might overcome these limitations are the following:• Development of the assay with direct labels—Avoid the use of a generic
reagent.• Reconfigure the assay with a larger volume—This allows further dilu-
tion of the substrate by addition of the detection reagents and reduces the requirement for the generic SA-XL665 reagent.
Summary
Time-resolved FRET assays enable homogeneous binding assays. One advantage of TR-FRET is that it places no particular size requirements on the binding pair, as does fluorescence polarization. A disadvantage is that it requires two labels.
Working with a model system affords the opportunity to get familiar with a potentially new chemistry and instrument parameters.
Prior to development of a TR-FRET assay for use in the HTS environment, follow these guidelines:
• Define assay performance specifications • Determine the required donor concentrations• Optimize donor/acceptor concentrations• Verify assay performance and specificity• Optimize other parameters such as enzyme assay conditions
References
Zhang, J., Chung, T.D.Y., Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays.
J. Biomol. Screen.
4:67–73.
Li, Z., Mehdi, S., Patel, I., Kawooya, J., Judkins, M., Zhang, W., Diener, K., Lozada, A., Dunnington, D. 2000. An ultra-high throughput screening approach for an adenine transferase using fluorescence polarization.
J. Biomol. Screen.
5:31–37.
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