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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 

 

 



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 



 

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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