LanthaScreen
™
Terbium-labeled Biotin Binding Reagents
User Guide
Catalog nos. PV3965, PV3966,
PV4749, PV4750
Shipping Condition: Dry Ice
Storage: –20°C
Protocol part no. PV396X.pps
Rev. date: 3 May 2007
For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760-603-7200 extension 40266
For information on frequently asked questions regarding the LanthaScreen
™
technology, please go to
www.invitrogen.com/lanthascreen
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
TABLE OF CONTENTS
1.
Reagents Available .................................................................................................................................................. 1
2.
Introduction .............................................................................................................................................................. 1
3.
Instrument Settings ................................................................................................................................................. 2
4.
Applications of Terbium-Labeled Biotin Binding Reagents to Protease Assays.............................................. 3
4.1
BACE1 Assay Using Tb-labeled Streptavidin ....................................................................................................................................3
4.2
BACE1 Assay Using Tb-Anti-Biotin Antibody ..................................................................................................................................4
5.
First-time Users........................................................................................................................................................ 5
6.
Assessing Data Quality in Ratiometric Measurements........................................................................................ 5
7.
Related Products...................................................................................................................................................... 6
8.
References................................................................................................................................................................ 7
9.
Notice to Purchaser ................................................................................................................................................. 7
1. REAGENTS
AVAILABLE
REAGENTS Size
Cat.
No.
50 µg
PV3965
LanthaScreen
™
Tb-Streptavidin, 1 mg/ml (17.9 µM)
1 mg
PV3966
25 µg
PV4749
LanthaScreen
™
Tb-Anti-Biotin antibody, 1 mg/ml (6.7 µM)
1 mg
PV4750
2. INTRODUCTION
For screening compound libraries, time-resolved fluorescence resonance energy transfer (TR-FRET) is a recognized
method for overcoming interference from compound autofluorescence or light scattering 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 is
brought within close proximity of one another, excitation of the first fluorophore (the donor) results 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 high throughput screening (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 because of 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) lasts a millisecond or longer. This contrasts sharply with the nanosecond-long
lifetimes of fluorophores commonly used in standard FRET assays. Because interference from autofluorescent compounds
or light scattering is also on the nanosecond timescale, these factors negatively affect standard FRET assays. TR-FRET
assays overcome these interferences 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 greatly reduces interference
Invitrogen • LanthaScreen
™
Terbium-labeled Biotin Binding Reagents User Guide
Page 2 of 7
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For information on frequently asked questions regarding the LanthaScreen
™
technology, please go to
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from background fluorescence or light scattering, but also avoids interference from direct excitation because of the non-
instantaneous nature of the flashlamp excitation source.
Although the terbium chelate donor directly attaches to a biomolecule such as a protein or peptide, it can also be
“indirectly” attached to a target molecule through a biotin-mediated association with terbium-labeled streptavidin or
anti-biotin antibody. Invitrogen’s terbium-labeled biotin binding reagents are therefore universal reagents that easily label
biotinylated biomolecules with terbium for use in TR-FRET assays. This guide discusses such a labeling strategy applied
to a protease assay using a peptide substrate that is labeled at one terminus with biotin and on the other with fluorescein.
3. INSTRUMENT
SETTINGS
The excitation and emission spectra of terbium and fluorescein are shown below 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. The exact specifications of
the excitation filter are not critical; filters with similar specifications will work well. In general, excitation filters that are
compatible with europium-based TR-FRET systems will perform well with the LanthaScreen
™
terbium chelates.
Figure 1:
Excitation and emission spectra of terbium and fluorescein.
As shown in the figure, four sharp peaks characterize the terbium emission spectrum, with silent regions between each
peak. The first terbium emission peak (between approximately 485 and 505 nm) overlaps the fluorescein maximum
excitation peak. The fluorescein maximum emission peak is centered in the silent region between the first and second
terbium emission peaks. Thus, energy transfer to fluorescein is measured in this region without interference from terbium
by using a filter centered at 520 nm with a 25-nm bandpass. The specifications of this filter are more critical than those of
the excitation filter. In general, standard “fluorescein-specific” filters will not perform well, because they also pass light
associated with the terbium emission spectrum. The emission of fluorescein because of FRET is referenced (or ratioed) to
the emission of the first terbium peak, using a filter that isolates this peak, typically 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 a 495-nm filter may be necessary with some instrument dichroic mirror choices (such as
those on the Tecan Ultra Evolution
™
instrument). 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. BMG Instruments offers a LanthaScreen
™
filter module for the BMG PHERAStar fluorescence plate
reader.
Aside from filter choices, instrument settings are similar to those used with europium-based technologies. In general, the
instrument manufacturer's guidelines serve as a good starting point for optimization. A LanthaScreen
™
assay would
Invitrogen •
LanthaScreen
™
Terbium-labeled Biotin Binding Reagents User Guide
Page 3 of 7
For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760-603-7200 extension 40266
For information on frequently asked questions regarding the LanthaScreen
™
technology, please go to
www.invitrogen.com/lanthascreen
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
typically use a delay time of 100 µs, followed by a 200-µs integration time. The number of flashes or measurements per
well is highly instrument-dependant and should be set as recommended by your instrument manufacturer. In general,
LanthaScreen
™
assays are run on any filter-based instrument capable of TR-FRET, such as the Tecan Ultra Evolution
™
,
BMG PHERAStar, Molecular Devices Analyst
®
, or PerkinElmer EnVision
™
fluorescence plate readers. LanthaScreen
™
assays have also been performed successfully on the Tecan Safire
2
monochromator-based instrument. Contact the
Invitrogen Corporation Technical Services group for instrument-specific setup guidelines.
4.
APPLICATIONS OF TERBIUM-LABELED BIOTIN BINDING REAGENTS TO PROTEASE ASSAYS
Terbium-labeled biotin binding reagents are useful as universal labeling reagents for biotinylated biomolecules such as
proteins, peptides, or oligonucleotides. In protease assays, Invitrogen’s terbium-based LanthaScreen
™
technology enables
TR-FRET protease assays to be performed using peptides that contain biotin- and fluorescein-modified termini. Such
peptides are readily prepared by standard peptide synthesis methods, and are inexpensively available from a variety of
vendors. The following describes the use of such reagents in an assay for β-secretase (BACE1) activity.
BACE1 is a key enzyme involved in the production of amyloid β-peptides found in the extracellular amyloid plaques of
Alzheimer's disease. In some cases, early-onset familial Alzheimer’s disease is attributed to a "Swedish" mutation in the
amyloid precursor protein (APP), which dramatically enhances cleavage of this protein by BACE1. This and other genetic
and pathological evidence led to therapeutic approaches that focus on inhibiting BACE1 and other APP-cleaving
enzymes.
To assay BACE1-catalyzed cleavage of the Swedish mutant of APP, a peptide corresponding to the cleavage site,
EVNL∧DAEF) (cleavage between residues L and D) was synthesized with an N-terminal biotin and a C-terminal
fluorescein. The fluorescein was attached to the ε-amino group of a lysine appended to the C-terminus of the peptide
sequence. This peptide was incubated with BACE1 in the presence or absence of an inhibitor for 1 hour at room
temperature, after which the reaction was stopped by adding Tb-SA in a pH 8.0 buffer (BACE1 is inactive at high pH) and
the plate read on a BMG PHERAStar fluorescence plate reader using the LanthaScreen
™
filter module available from
BMG. The assay is shown schematically below in Figure 2.
Figure 2:
Schematic of a LanthaScreen
™
protease assay using Tb-SA.
4.1
BACE1 Assay Using Tb-labeled Streptavidin
To determine the amount of BACE1 required for the assay, 100 nM of peptide substrate was incubated with a
dilution series of BACE1 in a 10-µL reaction volume of 50-mM sodium acetate buffer, pH 4.5. Each reaction was
performed in triplicate. After 1 hour, a 10-µL solution containing 10-nM Tb-SA and 300-mM Tris buffer, pH 8, in
TR-FRET dilution buffer (Invitrogen part number PV3574) was added. The high concentration of Tris buffer was
used to ensure that the final pH was slightly basic to stop the BACE1 reaction. After a short incubation
(approximately 10 minutes), the plate was read in TR-FRET mode on a Tecan Ultra Evolution
™
fluorescence plate
reader. The data are presented below in Figure 3A. Error bars are shown, but are extremely small.
Invitrogen • LanthaScreen
™
Terbium-labeled Biotin Binding Reagents User Guide
Page 4 of 7
For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760-603-7200 extension 40266
For information on frequently asked questions regarding the LanthaScreen
™
technology, please go to
www.invitrogen.com/lanthascreen
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
Figure 3:
(A) Titration of BACE1 against 100-nM peptide substrate. (B) Inhibition of
BACE1 by a statine containing peptide.
The enzyme titration experiment indicated that 0.1 units/mL BACE1 were appropriate for the assay. At this
enzyme concentration, approximately 50% of the substrate will be cleaved in the absence of inhibitor. The
inhibitor was a statine containing peptide (Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-(Statine)-Val-Ala-Glu-Phe-OH)
that was titrated against BACE1 in a two-fold dilution series beginning at 10 µM. Each reaction was performed in
triplicate. After 60 minutes, the reaction was stopped as described previously by the addition of Tb-SA and Tris
buffer, pH 8, to a final concentration of 5 nM Tb-SA and 150 mM Tris buffer. The plate was read on a Tecan Ultra
Evolution
™
fluorescence plate reader using standard instrument settings and the results are shown in Figure 3B.
Error bars are shown, but are extremely small. The statine-containing peptide inhibited BACE1 with an IC
50
value
of 32 nM, which agrees closely with the published value of 30 nM.
4.2
BACE1 Assay Using Tb-Anti-Biotin Antibody
To determine the amount of BACE1 required for the assay, 200 nM of peptide substrate was incubated with a
dilution series of BACE1 in a 15-µL reaction volume of 50-mM sodium acetate buffer, pH 4.5. Each reaction was
performed in triplicate. After 1 hour, a 5-µL solution containing 20-nM Tb-anti-Biotin antibody in LanthaScreen
™
BACE1 Assay Stop Solution was added. After a 1-hour incubation, the plate was read in TR-FRET mode on a
Tecan Ultra Evolution
™
fluorescence plate reader. The data are presented below in Figure 4A. Error bars are
shown, but are extremely small.
A. BACE1 Titration
B. BACE1 inhibitor 1
10
-4
10
-3
10
-2
10
-1
10
0
10
1
10
2
0.0
0.4
0.8
1.2
EC50 = 0.18 U/mL
BACE1 (U/mL)
E
m
is
si
o
n
R
a
ti
o
52
0:49
5
0.1
1
10
100
1000 10000 100000
0.0
0.2
0.4
0.6
0.8
1.0
1.2
IC
50
= 43.7 nM
BACE1 Inhibitor 1 (nM)
E
m
issi
o
n
R
at
io
520:
49
5
Figure 4:
(A) Titration of BACE1 against 200-nM peptide substrate. (B) Inhibition of
BACE1 by a statine-containing peptide.
Invitrogen •
LanthaScreen
™
Terbium-labeled Biotin Binding Reagents User Guide
Page 5 of 7
For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760-603-7200 extension 40266
For information on frequently asked questions regarding the LanthaScreen
™
technology, please go to
www.invitrogen.com/lanthascreen
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
The enzyme titration experiment indicated that 0.7 units/mL BACE1 were appropriate for the assay. At this
enzyme concentration, approximately 80% of the substrate will be cleaved in the absence of inhibitor. The
inhibitor was a statine containing peptide (Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-(Statine)-Val-Ala-Glu-Phe-OH)
that was titrated against BACE1 in a two-fold dilution series beginning at 10 µM. Each reaction was performed in
triplicate. After 60 minutes, the reaction was stopped as described previously by the addition of Tb-Anti-Biotin
Antibody in LanthaScreen
™
BACE1 Assay Stop Solution, to a final concentration of 5-nM Tb-anti-Biotin antibody.
The plate was read on a Tecan Ultra Evolution
™
fluorescence plate reader using standard instrument settings and
the results are shown in Figure 4B. Error bars are shown, but are extremely small.
5. FIRST-TIME
USERS
Invitrogen provides users with an aliquot of biotinylated, fluorescein labeled peptide (200 nM Fluorescein-Biotin Positive
Control in TR-FRET Dilution Buffer) as a positive control. To verify that instrument parameters are properly set for the
LanthaScreen
™
assay format, titrate a dilution series of the peptide against a fixed concentration of Tb-labeled reagents to
generate a binding curve. The certificate of analysis provided shows a binding curve representative of data expected from
such an experiment.
6.
ASSESSING DATA QUALITY IN RATIOMETRIC MEASUREMENTS
The TR-FRET ratio is a unitless value derived from the ratio of the underlying donor and acceptor signals. Because these
underlying signals depend 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 5 demonstrates the pitfalls of relying solely on the assay window to determine data quality. The ratiometric data in
Panel A is identical in quality (despite vastly different assay windows), as demonstrated in Panel B, after the data have
been normalized and re-plotted. The important factor in determining the robustness of an assay is the magnitude of errors
in the data compared to the difference between the maximum and minimum values, not the size of the instrument's assay
window. The Z´-factor value proposed by Zhang and colleagues (Zhang et al., 1999), which takes these factors into
account, offers a more accurate assessment of data quality in a TR-FRET assay. Typically, our assays have Z´-factor values
of greater than 0.70 (a Z´-factor value of 1 is the ideal; an assay exhibiting a Z´-factor value greater than 0.5 is considered
highly reliable).
Figure 5:
Assay window variability due to instrument type does not affect the
resulting data. A. Data obtained from three different instruments with different assay
windows. B. Upon normalization, the three curves in Panel A are identical.
Invitrogen • LanthaScreen
™
Terbium-labeled Biotin Binding Reagents User Guide
Page 6 of 7
For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760-603-7200 extension 40266
For information on frequently asked questions regarding the LanthaScreen
™
technology, please go to
www.invitrogen.com/lanthascreen
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
7. RELATED
PRODUCTS
REAGENTS
Volume 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 Substrate) Antibody
1 mg
PV3561
25 µg
PV3562
LanthaScreen
™
Tb-IκB 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
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
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 (YIYGSFK), 1 mg/mL
1 mg
PV3513
Fluorescein-PTK Substrate 2 (CDC2
(6-20)
), 1 mg/mL
1 mg
PV3511
Fluorescein-Poly GT, 30 µM
1 mL
PV3610
Fluorescein-Poly GAT, 30 µM
1 mL
PV3611
10 µg
PV3580
100 µg
PV3579
LanthaScreen
™
Thiol Reactive Tb Chelate
1 mg
PV3578
10 µg
PV3583
100 µg
PV3582
LanthaScreen
™
Amine Reactive Tb Chelate
1 mg
PV3581
Invitrogen •
LanthaScreen
™
Terbium-labeled Biotin Binding Reagents User Guide
Page 7 of 7
For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760-603-7200 extension 40266
For information on frequently asked questions regarding the LanthaScreen
™
technology, please go to
www.invitrogen.com/lanthascreen
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com
8. REFERENCES
Zhang, J. H., Chung, T. D., and 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
9.
NOTICE TO PURCHASER
Limited Use Label License No. 176: Lanthanide Chelates
This product is the subject of one or more U.S. Patents (patent nos. 5,622,821 5,639,615, 5,656,433, and 4,822,733) 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 Corporation is willing to accept return of the product for 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.
The performance of this product is guaranteed for six months from the date of purchase, if stored and handled properly.
Ultra Evolution
™
is a trademark of Tecan Group Ltd.
Analyst
™
is a trademark of Molecular Devices Corporation
EnVision
™
is a trademark of PerkinElmer
®
Life and Analytical Sciences
©2005–2007 Invitrogen Corporation. All rights reserved. For research use only.
Not intended for any animal or human therapeutic or diagnostic use.
Document Outline - 1. REAGENTS AVAILABLE
- 2. INTRODUCTION
- 3. INSTRUMENT SETTINGS
- 4. APPLICATIONS OF TERBIUM-LABELED BIOTIN BINDING REAGENTS T
- 4.1 BACE1 Assay Using Tb-labeled Streptavidin
- 4.2 BACE1 Assay Using Tb-Anti-Biotin Antibody
- 5. FIRST-TIME USERS
- 6. ASSESSING DATA QUALITY IN RATIOMETRIC MEASUREMENTS
- 7. RELATED PRODUCTS
- 8. REFERENCES
- 9. NOTICE TO PURCHASER
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