LanthaScreen™ Terbium Labeled TR-FRET
Secondary Antibody Reagents-User Guide
Cat. nos. PV3765, PV3767, PV3769, PV3771, PV3773, PV3775, PV3777, and PV3779
O-062132-r1 US 0405
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
TABLE OF CONTENTS
1.0
REAGENTS AVAILABLE............................................................................................................................................................................... 1
2.0
INTRODUCTION............................................................................................................................................................................................. 1
3.0
INSTRUMENT SETTINGS ............................................................................................................................................................................ 2
4.0
APPLICATIONS OF ANTI-SPECIES ANTIBODIES TO KINASE ASSAYS ..................................................................................... 2
5.0
FIRST-TIME USERS ........................................................................................................................................................................................ 4
6.0
ASSESSING DATA QUALITY IN RATIOMETRIC MEASUREMENTS ........................................................................................... 4
7.0
RELATED PRODUCTS ................................................................................................................................................................................... 5
8.0
NOTICE TO PURCHASER............................................................................................................................................................................. 6
1.0 REAGENTS
AVAILABLE
Reagents Size
Cat.
no.
25 µg
PV3765
LanthaScreen™ Tb-anti-Mouse Antibody
1 mg
PV3767
25 µg
PV3769
LanthaScreen™ Tb-anti-Goat Antibody
1 mg
PV3771
25 µg
PV3773
LanthaScreen™ Tb-anti-Rabbit Antibody
1 mg
PV3775
25 µg
PV3777
LanthaScreen™ Tb-anti-Human Antibody
1 mg
PV3779
2.0 INTRODUCTION
When screening libraries of compounds, time-resolved FRET (TR-FRET) is a recognized method for overcoming interference from
compound autofluorescence or light scatter from precipitated compounds. The premise of a TR-FRET assay is the same as that of a
standard FRET assay: when a suitable pair of fluorophores are brought within close proximity of one another, excitation of the first
fluorophore (the donor) can result in energy transfer to the second fluorophore (the acceptor). This energy transfer is detected by an
increase in the fluorescence emission of the acceptor, and a decrease in the fluorescence emission of the donor. In HTS assays, FRET is
often expressed as a ratio of the intensities of the acceptor and donor fluorophores. The ratiometric nature of such a value corrects for
differences in assay volumes between wells, and corrects for quenching effects due to colored compounds.
In contrast to standard FRET assays, TR-FRET assays use a long-lifetime lanthanide chelate as the donor species. Lanthanide chelates
are unique in that their excited state lifetime (the average time that the molecule spends in the excited state after accepting a photon)
can be on the order of a millisecond or longer. This is in sharp contrast to the lifetime of common fluorophores used in standard FRET
assays, which are typically in the nanosecond range. Because interference from autofluorescent compounds or scattered light is also
on the nanosecond timescale, these factors can negatively impact standard FRET assays. To overcome these interferences, TR-FRET
assays are performed by measuring FRET after a suitable delay, typically 50 to 100 microseconds after excitation by a flashlamp
excitation source in a microtiter plate reader. This delay not only overcomes interference from background fluorescence or light
scatter, but also avoids interference from direct excitation due to the non-instantaneous nature of the flashlamp excitation source.
The most common lanthanides used in TR-FRET assays for HTS are terbium and europium. Terbium offers unique advantages over
europium when used as the donor species in a TR-FRET assay. In contrast to europium based systems that employ APC as the
acceptor, terbium-based TR-FRET assays can use common fluorophores such as fluorescein as the acceptor. Because it is
straightforward (and inexpensive) to label a molecule such as a peptide with fluorescein, directly labeled biomolecules may be used
in terbium-based TR-FRET assays, rather than biotinylated molecules that must then be indirectly labeled via streptavidin-mediated
recruitment of APC. The use of directly labeled molecules in a terbium-based TR-FRET assay reduces costs, improves kinetics, avoids
problems due to steric interactions involving large APC conjugates, and simplifies assay development, since there are fewer
independent variables requiring optimization in a directly labeled system.
Invitrogen • LanthaScreen™ Terbium Labeled TR-FRET Secondary Antibody Reagents-User Guide • O-062132-r1 US 0405
Page 2 of 6
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
3.0 INSTRUMENT
SETTINGS
The excitation and emission spectra of terbium and fluorescein are shown in Figure 1. As with other TR-FRET systems, the terbium
donor is excited using a 340 nM excitation filter with a 30 nm bandpass. However, the exact specifications of the excitation filter are
not critical, and filters with similar specifications will work well. In general, excitation filters that work with europium-based TR-
FRET systems will perform well with the LanthaScreen™ terbium chelates.
As is shown in the figure, the terbium emission spectrum is characterized by four sharp emission peaks, with silent regions between
each peak. The first terbium emission peak (centered between approximately 485 and 505 nm) overlaps with the maximum excitation
peak of fluorescein. Energy transfer to fluorescein is then measured in the silent region between the first two terbium emission peaks.
Because it is important to measure energy transfer to fluorescein without interference from terbium, a filter centered at 520 nm with a
25 nm bandpass is used for this purpose. The specifications of this filter are more critical than those of the excitation filter. In general,
standard “fluorescein” filters may not be used, because such filters also pass light associated with the terbium spectra as well. The
emission of fluorescein due to FRET is referenced (or “ratioed”) to the emission of the first terbium peak, using a filter that isolates
this peak. This is typically accomplished with a filter centered at 490 or 495 nm, with a 10 nm bandpass. In general, a 490 nm filter will
reduce the amount of fluorescein emission that “bleeds through” into this measurement, although instrument dichroic mirror choices
(such as those on the Tecan Ultra instrument) may necessitate the use of a 495 nm filter. The effect on the quality of the resulting
measurements is minimal in either case. Filters suitable for LanthaScreen™ assays are available from Chroma (www.chroma.com) as
filter set PV001, or from other vendors. A LanthaScreen™ filter module for the BMG PheraStar is available direct from BMG
Instruments.
300
350
400
450
500
550
600
650
0
10
20
30
40
50
60
70
80
90
100
110
Wavelength (nm)
Excitation/Emission
Fluorescein
Terbium
Figure 1—
Excitation and Emission spectra of fluorescein and terbium.
Aside from filter choices, instrument settings are typical to the settings used with europium-based technologies. In general,
guidelines provided by the instrument manufacturer can be used as a starting point for optimization. A delay time of 100 µs,
followed by a 200 µs integration time, would be typical for a LanthaScreen™ assay. The number of flashes or measurements
per well is highly instrument dependant and should be set as advised by your instrument manufacturer. In general,
LanthaScreen™ assays can be run on any filter-based instrument capable of time-resolved FRET, such as the Tecan Ultra,
BMG PheraStar, Molecular Devices Analyst, or PE Envision. LanthaScreen™ assays have also been performed successfully on
the Tecan Safire
2
monochromator-based instrument. Contact Invitrogen Technical Services for instrument-specific setup
guidelines.
4.0
APPLICATIONS OF ANTI-SPECIES ANTIBODIES TO KINASE ASSAYS
LanthaScreen™ assays may be performed with reagents that are directly labeled with a terbium chelate, or by using reagents
that are “indirectly” labeled through association with another moiety that is itself labeled with a terbium chelate. For example,
in antibody-based kinase assays, one can utilize a phosphospecific antibody that is directly labeled with terbium, or an
unlabeled phosphospecific primary antibody that is then “indirectly” labeled with terbium through association with a
terbium-labeled secondary antibody that specifically binds to the phosphospecific primary antibody. Although this strategy is
not as straightforward as using a directly labeled primary antibody, it can be advantageous when the directly labeled primary
antibody is not readily available. For example, labeling of a primary antibody typically requires larger amounts of purified,
concentrated antibody that may be expensive or time-consuming to obtain. The suitability of such an antibody in a particular
assay application can be readily evaluated through the use of a Tb-labeled secondary antibody using the approach described
below. Depending on the results of such an evaluation, one may then decide whether or not to examine such an assay using a
directly labeled antibody.
Invitrogen • LanthaScreen™ Terbium Labeled TR-FRET Secondary Antibody Reagents-User Guide • O-062132-r1 US 0405
Page 3 of 6
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
The principle of a LanthaScreen™ kinase assay using a Tb-labeled secondary antibody is shown below in Figure 2. The assay
itself can be divided into three phases: the kinase reaction phase, primary antibody incubation phase, and the detection phase.
In the kinase reaction phase, all components required for the kinase reaction are added to the well, including fluorescein
labeled substrate, and the kinase reaction is allowed to incubate for a set period of time, typically 60 to 90 minutes. After the
reaction, EDTA is added to stop the kinase reaction and the primary anti-phosphospecific antibody is added to bind
phosphorylated product. The incubation is conducted for 30 minutes after which the Tb-labeled secondary antibody is added
to detect the complex. Binding of the Tb-labeled secondary antibody to the primary antibody that is associated with the
fluorescein-labeled phosphorylated product brings the terbium and fluorescein into close proximity, resulting in an increase in
TR-FRET. In the presence of an inhibitor, formation of phosphorylated product is reduced, and the TR-FRET value is
decreased.
Figure 2—
Schematic illustration of the use of a terbium labeled secondary antibody in a kinase assay format.
There are a range of variables associated with developing a TR-FRET assay utilizing Tb-labeled secondary antibodies.
Optimized reagent concentrations, order of antibody additions (i.e. primary first, secondary first, or premixed primary and
secondary), incubation times, etc., must be determined by the end user for a particular application.
4.1
Antibody concentration determination
It is also important to determine the optimal concentration of secondary antibody to be used in the assay. While too little can
cause a lack of appreciable signal generation, too much can increase the background such that dynamic range can become
compromised. Too much or too little secondary antibody may result in a smaller “read window”. We recommend determining
the optimal concentration of the Tb-labeled secondary by using the secondary antibody at 0.5X, 1X, 2X, and 4X concentrations
of the primary antibody. From an experiment such as this, a user can balance the size of the read window versus the amount
of antibody used in order to determine optimal amounts of secondary antibody for their particular application. The optimal
amount of secondary antibody used will depend on the amount of primary antibody used, and would need to be determined
for a given concentration of primary antibody used in the assay.
4.2
Assay stability and read window
For a given assay system, signal stability and read window should be assessed. In general, many assays will reach equilibrium
within 2 hours, and will show a stable signal for at least 6 hours. However, depending on assay configuration and the specific
demands of the assay, these times may vary and should be determined experimentally for the given assay system.
4.3 Cross-reactivity
In all cases, the species-specific antibodies bind to their corresponding species IgG with low nM apparent K
d
’s, and show
negligible cross reactivity with IgG's from other species. The Tb-anti-Goat antibody exhibits this highest level of cross-
reactivity with the Fluorescein-Human IgG, but this is minor in comparison to the signal from its corresponding IgG (data not
shown).
Additionally, the Tb-anti-Mouse antibody has been demonstrated to react with Fluorescein-Mouse IgM antibody for use in
assays that employ mouse antibodies of the IgM isotype (data not shown).
Invitrogen • LanthaScreen™ Terbium Labeled TR-FRET Secondary Antibody Reagents-User Guide • O-062132-r1 US 0405
Page 4 of 6
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
5.0
FIRST TIME USERS
Each Tb-labeled anti-species antibody from Invitrogen is provided with a corresponding fluorescein-labeled IgG as a positive
control. To verify that instrument parameters are properly set-up to run the LanthaScreen™ assay format, it is suggested that
a dilution series of the fluorescein-labeled IgG should be titrated against a fixed concentration of Tb-labeled antibody in order
to generate a binding curve. The certificate of analysis provided with each antibody kit contains a binding curve that is
representative of the data that should be obtained in conducting such an experiment. As an example, Figure 3 shows the
results of a titration series of Fluorescein-Mouse IgG against 2 nM Tb-labeled Anti-Mouse Antibody. The plate was read on a
Tecan Ultra plate reader after a 2 hour room temperature incubation.
0.001
0.01
0.1
1
10
100
0.0
0.5
1.0
1.5
Fluorescein-IgG (nM)
TRFRET Ratio
Figure 3—
Titration of Fluorescein-Mouse IgG into Tb-Anti-Mouse antibody on the Tecan Ultra.
6.0
ASSESSING DATA QUALITY IN RATIOMETRIC MEASUREMENTS
The TR-FRET value is a unitless ratio derived from the underlying donor and acceptor signals. Because the underlying donor
and acceptor signals are dependant on instrument settings (such as instrument gain), the TR-FRET ratio, and the resulting
“top” and “bottom” of an assay window will depend on these settings as well, and will vary from instrument to instrument.
Figure 4 is instructive in demonstrating the pitfalls of simply relying on the assay window as a measure of data quality. The
ratiometric data on the left is all identical in quality (despite vastly different assay windows), as is evident when the curves are
normalized and re-plotted in the graph at right. What is important in determining the robustness of an assay is not the size of
the window as much as the size of the errors in the data relative to the difference in the maximum and minimum values. It is
for this reason that the “Z prime” value proposed by Zhang and colleagues (J Biomol Screen 1999: 4(2) pp 67-73), which takes
these factors into account, is the correct way to assess data quality in a TR-FRET assay. Typically, our assays have Z’ values of
greater than 0.70.
0.001
0.01
0.1
1
10
100
0.00
0.25
0.50
0.75
1.00
1.25
[Tracer] (nM)
0.001
0.01
0.1
1
10
100
[Tracer] (nM)
520:495 Ratio
0
25
50
75
100
% TR-FRET
Instrument 1
Instrument 2
Instrument 3
Instrument 1
Instrument 2
Instrument 3
A
B
Figure 4—
Assay window variability due to instrument type does not affect the resulting data and upon normalization, the above three curves
are identical.
Invitrogen • LanthaScreen™ Terbium Labeled TR-FRET Secondary Antibody Reagents-User Guide • O-062132-r1 US 0405
Page 5 of 6
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
7.0 RELATED
PRODUCTS
Reagents Size
Cat.
no.
25 µg
PV3552
LanthaScreen™ Tb-PY20 Antibody
1 mg
PV3553
25 µg
PV3554
LanthaScreen™ Tb-PY72 Antibody
1 mg
PV3555
25 µg
PV3556
LanthaScreen™ Tb-PY100 Antibody
1 mg
PV3557
25 µg
PV3558
LanthaScreen™ Tb-PT66 Antibody
1 mg
PV3559
25 µg
PV3560
LanthaScreen™ Tb-pSer (PKC) Antibody
1 mg
PV3561
25 µg
PV3562
LanthaScreen™ Tb-I
κBα pSer32 Antibody
1 mg
PV3563
25 µg
PV3564
LanthaScreen™ Tb-pCrosstide Antibody
1 mg
PV3565
25 µg
PV3566
LanthaScreen™ Tb-CREB pSer133 Antibody
1 mg
PV3567
50 µg
PV3965
LanthaScreen™ Tb-Streptavidin, 1 mg/ml
1 mg
PV3966
Fluorescein-PKC Substrate, 1 mg/ml
1 mg
PV3506
Fluorescein-IKK Substrate, 1 mg/ml
1 mg
PV3507
Fluorescein-CREBtide Substrate, 1 mg/ml
1 mg
PV3508
Fluorescein-Crosstide Substrate, 1 mg/ml
1 mg
PV3509
Fluorescein-PTK Substrate 1, 1 mg/ml
1 mg
PV3513
Fluorescein-PTK Substrate 2, 1 mg/ml
1 mg
PV3511
Fluorescein-Poly GT, 30 µM
1 ml
PV3610
Fluorescein-Poly GAT, 30 µM
1 ml
PV3611
10 µg
PV3583
100 µg
PV3582
LanthaScreen™ Amine Reactive Tb Chelate
1 mg
PV3581
Invitrogen • LanthaScreen™ Terbium Labeled TR-FRET Secondary Antibody Reagents-User Guide • O-062132-r1 US 0405
Page 6 of 6
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
8.0
NOTICE TO PURCHASER
Limited Use Label License No. 176: Lanthanide Chelates
This product is the subject of one or more of US Patents 5,622,821 5,639,615, and 5,656,433 and foreign equivalents. The
purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and
components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The
buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its
components to a third party or otherwise use this product or its components or materials made using this product or its
components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product
to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in
writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information
solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration
and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its
components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or
prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are
resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above
patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product
developed in research by the buyer in which this product or its components was employed, provided that neither this product
nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the
limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For
information on purchasing a license to this product for purposes other than research, contact Licensing Department,
Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.
Limited Use Label License No. 178: Lifetime-resolved assay procedures
This product is sold under license to U.S. Patent 4,822,733 from Vysis, an Abbott Laboratories company. This product is
licensed for research use only and may not be used for in vitro diagnostics.
LanthaScreen™ is a trademark of Invitrogen Corporation.
©2005 Invitrogen Corporation. All rights reserved. Reproduction forbidden without permission.
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