Application Note - Invitrogen

Application Note 

Direct LanthaScreen Kinase Assays: 

Applications on the BMG LABTECH PHERAstar 

Randy Hoff man, Megan Buros, and Kevin Kupcho 

Invitrogen Corporation • 501 Charmany Drive • Madison, Wisconsin • 53719 USA 

Abstract 

TR-FRET assays have traditionally used europium as the long-lifetime donor label 

and the fluorescent protein allophycocyanin (also known as APC or XL-665) as the 

acceptor species. Due to its size (>100 kD), APC is typically used as a streptavidin 

conjugate to indirectly label a biotinylated substrate. In contrast, the LanthaScreen 

TR-FRET platform from Invitrogen Drug Discovery Solutions uses terbium in place of 

europium as the long-lifetime donor species, and fluorescein as the acceptor species. 

The terbium-based LanthaScreen configuration has several advantages over the tri- 

molecular donor/biotinylated substrate/streptavidin-APC format. These include sim- 

pler assay optimization, faster kinetics of complex formation, and avoidance of steric 

problems associated with the large streptavidin-APC moiety, as well as the cost and 

lot-to-lot consistency of fluorescein relative to streptavidin-APC. We have successfully 

applied this technology to a variety of target classes such as kinases and proteases, 

and we have demonstrated the resistance of the readout format to interference from 

color quenchers, light scatterants, or fluorescent compounds. This application note 

focuses on kinase applications. 

Toll Free: 800 955 6288 • E-mail: tech_service@invitrogen.com • www.invitrogen.com


Kinases and disease 

Protein kinases (PKs) are a diverse group of enzymes involved in many 

areas of cell signaling. These include cell growth and proliferation as 

well as neural functions. The keen interest in PKs arises from their role in 

regulating biological mechanisms. Through phosphorylation, PKs participate 

in many cellular signal transduction processes. Furthermore, 

defects in these pathways have been implicated in numerous human 

diseases including cancer, inflammation, and diabetes. Research 

focused on kinase activity provides the means to identify targets that 

can be used to develop new pharmaceutical agents for treatment of 

many of these diseases. 

Advantages of TR-FRET 

Time Resolved Fluorescence (TR-FRET) assays offer advantages over 

fluorescence polarization (FP) and fluorescence intensity (FI) assays when 

background fluorescence is a problem. Because the donor species used in 

a TR-FRET assay has a fluorescent lifetime that is many orders of magnitude 

longer than background fluorescence or scattered light, energy transfer 

can be measured after the interfering background signal has completely 

decayed. Additionally, the FRET nature of the assay allows ratiometric data 

analysis, eliminating well-to-well variations often seen in FI assays. 

Why terbium? 

Although europium is the most commonly used donor in TR-FRET assays, 

terbium offers the advantage of using either fluorescein or rhodamine 

as the acceptor. Any FP or FI assay that is based on a fluorescein- or rhodamine-labeled 

ligand binding to a receptor can therefore be converted 

to a TR-FRET assay by labeling the protein partner with a terbium chelate. 

The background fluorescence effects sometimes associated with 

fluorescein are eliminated because the assays are time resolved. 

Because the donor-acceptor pair in a terbium-based TR-FRET assay 

does not require a biotin-avidin mediated interaction—as is required 

for most europium-based assays—optimization of reagents is simpler. 

Many europium-based assays use the interaction of biotin with streptavidin-APC. 

Because of the large size of this complex, steric hindrance 

can complicate assay development. Fluorescein is small in comparison 

(does not cause steric hindrance) and is soluble compared to some of 

the “sticky” far-red dyes. 

Figure 2—Direct LanthaScreen TR-FRET kinase assay schematic 

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Figure 1—BMG LABTECH PHERAstar 

The PHERAstar is the new multifunctional microplate reader from BMG LABTECH developed 

to meet the challenges of HTS. 

The PHERAstar: ideal for LanthaScreen assays 

BMG LABTECH, a global manufacturer of microplate measurement 

and handling systems for basic research and high throughput screening 

(HTS), has recently launched the new PHERAstar multifunctional 

microplate reader (Figure 1). The PHERAstar is designed to provide significant 

improvements in sensitivity and read times for all plate formats 

up to 1536-well and in all leading non-radioactive detection modes: 

fluorescence intensity (including FRET), time-resolved fluorescence 

(including LanthaScreen), high-end fluorescence polarization (including 

PolarScreen), luminescence (including BRET), and absorbance 

(UV/VIS). The PHERAstar utilizes a unique application-specific module 

design in conjunction with an optical reading head featuring five 

photomultiplier tubes for simultaneous dual emission at any desired 

wavelength. This optical design provides for outstanding sensitivity 

and accuracy in fluorescence- and luminescence-based assays. The 

simultaneous measurement minimizes the read time for new, sophisticated 

applications like LanthaScreen kinase assays. 

Direct LanthaScreen format 

Because LanthaScreen assays are available as a direct format (i.e., 

non-competitive), customers can design an assay to their specifications 

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A fluorescein-labeled kinase substrate peptide is incubated with kinase and ATP. Terbium-labeled antibody is then added and phosphorylation detected by an increase in the TR-FRET ratio. 

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using reagents from Invitrogen’s LanthaScreen Toolbox. Components 

of the toolbox include complete reagent sets (Tb-labeled antibodies 

and fluorescein-labeled substrates) to assay a broad range of kinases. 

In addition, “generic” reagents such as streptavidin are included to 

facilitate assay development against a range of diverse target classes. 

Custom labeling services are also available to provide solutions to assay 

problems. See Figure 2 for the assay mechanism. 

How important is instrumentation? 

The instrumentation requirements for a terbium-based LanthaScreen 

assay are minimal. The keys are (1) having the proper optical specifications 

(including filters and dichroics) and (2) understanding the 

software settings needed for the assay. BMG LABTECH makes the task 

especially easy for users by providing application-specific optical modules. 

For example, the optical module used in these experiments, the 

LanthaScreen module, has been pre-qualified by BMG LABTECH and is 

available directly from the company. Figure 3 shows plate reader setup 

on the BMG LABTECH PHERAstar instrument. 

Figure 3—Plate reader setup 

This screenshot shows an assay setup window from the PHERAstar. A LanthaScreen optical 

module (including filters and dichroics) is available for the PHERAstar directly from BMG 

LABTECH. 

Table 1—Direct LanthaScreen on the PHERAstar: Statistics for PKC isoform titrations 

Protocol for PKC isoforms 

To demonstrate the functionality of Direct LanthaScreen technology on 

the PHERAstar, multiple PKC isoforms were titrated to determine optimal 

kinase concentrations for screening. The following protocol is an example 

of the conditions used to determine the EC₅₀ for one of the isoforms, PKCα. 

PKCα titration with Direct LanthaScreen technology 

A dilution series of PKCα, starting at a final concentration of 2.0 µg/ml, 

was incubated in the presence of 250 nM fluorescein-labeled PKC substrate 

and 20 µM ATP in a total volume of 10 µl in a black Corning® lowvolume 

384-well plate (Corning #3676). After a 90-minute incubation 

at room temperature, 10 µl of TR-FRET dilution buffer containing 2X 

EDTA (20 mM) and 2X Tb-PKC antibody (1.0 nM) was added and mixed 

to create a final volume of 20 µl per well, a final 1X EDTA concentration 

of 10 mM, and a final 1X antibody concentration of 0.5 nM. After a 60minute 

incubation at room temperature, the plate was read on the BMG 

LABTECH PHERAstar. Each data point represents the average of three 

wells. Figure 4 shows representative kinase titration curves for PKCα 

and PKCζ; Table 1 shows statistical data for all ten kinases tested. 

Figure 4—Direct LanthaScreen on the PHERAstar: PKCα and ζ titrations 

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PKCα PKCβI PKCβII PKCδ PKCε PKCη PKCγ PKCι PKCθ PKCς 

EC 50 (ng/ml) 0.43 0.27 0.22 0.07 0.03 0.04 0.18 0.13 0.16 0.12 

Min. value 0.44 0.43 0.79 .55 0.73 0.57 0.47 0.52 1.02 0.84 

Max. value 2.69 2.66 2.61 2.64 2.63 2.65 2.64 2.67 2.63 2.63 

Max-min 2.25 2.23 1.82 2.09 1.90 2.08 2.17 2.15 1.61 1.79 

Fold difference (max/min) 6.1 6.2 3.3 4.8 3.6 4.6 5.6 5.1 2.6 3.1 

Hill slope 1.1 0.89 1.01 0.70 0.93 1.10 0.87 0.73 0.96 0.83 

R² 0.991 0.996 0.988 0.979 0.988 0.993 0.994 0.988 0.976 0.982 

S:N 60 47 18 31 25 32 42 46 14 18 

Z’-factor value 0.94 0.91 0.78 0.87 0.85 0.88 0.91 0.91 0.74 0.79 

All ten PKC isoforms were titrated in a similar manner to the method described for PKCα. This data is meant to familiarize the reader with the relationship between Z’-factor values and other common 

statistical parameters. Regardless of “fold difference” or signal-to-noise ratios, all assays provided Z’-factor values far greater than 0.5. This is due to the use of FRET-based, ratiometric data analysis, 

which leads to low standard deviations of the replicates. Both Hill slope and R² measurements fall within acceptable limits. A Hill slope of 1.0 indicates the ideal binding curve; R² values approaching 

1.0 indicate the perfect curve fit. 

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Ten PKC isoforms were tested for optimal kinase concentration in the Direct LanthaScreen 

format. This figure shows two representatives, alpha and zeta. Both kinases have EC₅₀ concentrations 

in the sub ng/ml range. See Table 1 for statistical details on all 10 kinases.

Data analysis 

Signal-to-noise ratio is calculated using the following formula: 

where µ p = mean of “positive control” (min ratio), µ n = mean of “negative 

control” (max ratio), and σ = the corresponding standard deviations. 

Z’-factor value is calculated using the following formula: 

where µ p = mean of “positive control” (min ratio), µ n = mean of “negative 

control” (max ratio), and σ = the corresponding standard deviations. 

Protocol for universal tyrosine kinase assay 

To demonstrate the functionality of a universal tyrosine kinase assay 

using Direct LanthaScreen, both fluorescein-poly-GAT (Glu, Ala, 

Tyr) and fluorescein-poly-GT (Glu, Tyr) substrates were used with terbium-labeled 

PY20 antibody. Figure 5 shows titration curves for two 

TK kinases using both substrates. Table 2 shows the EC₅₀ calculations 

derived from kinase titrations using both substrates. 

A dilution series of kinase was incubated with 400 nM fluoresceinlabeled 

substrate and 200 µM ATP in a total volume of 10 µl in a black 

Corning® low-volume 384-well plate (Corning #3676). After a 60-minute 

incubation at room temperature, 5 µl of TR-FRET dilution buffer 

containing 4X EDTA (60 mM) was added. Next, 5 µl of TR-FRET dilution 

buffer containing 4X antibody (8 nM) was added and mixed to create a 

final volume of 20 µl per well, a final 1X EDTA concentration of 15 mM, 

and a final 1X antibody concentration of 2 nM. After a 60-minute incubation 

at room temperature, the plate was read on the BMG LABTECH 

PHERAstar. Each data point represents the average of four wells. 

Figure 5—Kinase titrations for ZAP-70 and IGF-1R using both TK substrates 

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Table 2—EC₅₀ calculations from kinase titration assays using either poly- 

GAT or poly-GT substrate 

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A total of 24 tyrosine kinases were titrated in the presence of 200 µM ATP and either fluorescein-poly-GAT (Glu, Ala, Tyr) or fluorescein-poly-GT (Glu, Tyr) substrate. This figure shows representative 

results for ZAP-70 (A and B) and IGF-1R (C and D). As a trend, poly-GAT reactions yielded higher dynamic ranges, whereas poly-GT reactions required less kinase as determined by EC₅₀ calculations. 

Kinase 

EC₅₀ (ng/ml) 

EC₅₀ (ng/ml) 

Kinase 

polyGAT polyGT polyGAT polyGT 

ZAP-70 7.46 1.98 LynA 0.38 0.34 

TEC 5.02 14.70 LynB 1.01 0.08 

ErbB2 9.16 3.41 Abl1 4.73 0.39 

cKit 5.22 6.01 Arg 4.70 0.54 

cMet 1.25 0.71 FGFR1 0.45 0.29 

IGF1R 15.54 0.57 FGFR2 0.42 0.06 

FLT3 0.66 0.02 FGFR3 0.64 0.03 

PDGFRβ 3.40 1.84 FGFR4 0.97 0.02 

Lck 1.06 2.64 EGFR 15.55 9.74 

Fyn 7.48 0.53 CSF1R 0.37 0.30 

Yes 6.94 0.56 TrkA 0.08 0.10 

Src 0.75 0.16 EphB4 0.36 0.51 

Dynamic range: poly-GAT ~ 50 fold 

Dynamic range: poly-GT ~ 17 fold 

This table shows the trend for lower kinase concentrations needed (as shown by EC₅₀ values) 

using the poly-GT substrate. Note, however, that the poly-GT substrate generally leads to 

increased assay dynamic range.


Results and conclusions 

Our results for the PKC isoform titrations demonstrate that Direct 

LanthaScreen PKC assays are sensitive in that they require sub ng/ml 

kinase concentrations. This is an important factor in keeping overall 

screening costs down. In addition, the assays are robust with Z’-factor 

values greater that 0.7 in all cases. These Z’-factor values provide confidence 

that the assay data is meaningful. It is important to stress this 

point: “Fold difference” (dynamic range, assay window) is not critical to 

assay robustness. Rather, the difference between positive and negative 

controls, combined with standard deviations of the same, lead to assay 

confidence as dictated by Z’-factor values. 

Our results for the TK assays show that use of a fluorescein-labeled 

substrate common for many TKs (poly-GT or poly-GAT) allows development 

of a “universal” TK assay using the Direct LanthaScreen format. 

For many of the TKs shown, EC 50 values are similar for both substrates. 

But for a number of kinases, especially the FGFRs, EC₅₀ values are much 

lower for the poly-GT substrate. This may indicate that the poly-GT 

substrate provides a more sensitive assay compared to the poly-GAT. 

However, on average, the poly-GAT substrate provided an increased 

dynamic range compared to poly-GT (~50 fold compared to ~17 fold). 

Therefore, depending on the kinase, the customer can choose which is 

more important: using less kinase or having increased dynamic range. 

Ordering information 

The terbium-based Direct LanthaScreen format offers several advantages 

over many other fluorescent assays, including traditional europium-based 

formats: 

1. The ratiometric nature of LanthaScreen eliminates well-to-well 

data variation. 

2. The time-resolved nature of LanthaScreen assays allows for the 

use of fluorescein without the associated drawbacks of compound 

interference. 

3. The ability to use fluorescein as an acceptor rather than APC simplifies 

assay development and reduces cost. 

4. Steric hindrance due to the biotin-streptavidin:APC complex is 

eliminated when using the relatively small fluorescein molecule. 

5. Fluorescein is very soluble, unlike some “stickier” red-shifted dyes. 

6. It is approximately 5–10 fold less expensive to label a peptide substrate 

with fluorescein than with a far-red dye such as Cy5. 

From an instrumentation point of view, BMG LABTECH provides an 

excellent platform to simplify LanthaScreen assay development. The 

PHERAstar provides the speed and sensitivity to truly take advantages 

of either LanthaScreen format described above. An optical module 

optimized for use with LanthaScreen assays is available from BMG 

LABTECH, making assay setup easy. 

Product Quantity Cat. no. 

Fluorescein PKC substrate, 1.0 mg/ml 1 mg PV3506 

Kinase Buffer, 5X (for PKC assays) 4 ml PV3189 

Kinase Buffer (for TK assays) 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 

5 mM MnCl₂, 2 mM DTT, 200 µM Na₃VO₄, and 0.01% CHAPS 

LanthaScreen Tb-PKC Substrate Antibody 

N/A N/A 

25 µg PV3536 

100 µg PV3537 

PKCα 5 µg P2232 

LanthaScreen TR-FRET Dilution Buffer 200 ml PV3574 

ZAP-70 20 µg P2782 

LanthaScreen Tb-PY20 Antibody 

25 µg PV3528 

1 mg PV3529 

Fluorescein-poly-GAT (Glu, Ala, Tyr) 1 mg PV3611 

Fluorescein-poly-GT (Glu, Tyr) 1 mg PV3610 

Kinase Quench Buffer (500 mM EDTA) 1 ml P2825


These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or www.invitrogen.com). 

By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. 

For research use only. Not intended for any animal or human therapeutic or diagnostic use. 

©2004 Invitrogen Corporation. All rights reserved. Reproduction forbidden without permission. Printed in the U.S.A. 

Corporate headquarters: 

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O-13473-r1 US 1204

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