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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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