The Adapta™ universal kinase assay:
A superior alternative to luciferase-based kinase assays
Kevin Kupcho, Deborah K. Stafslien, Upinder Singh, Hildegard C. Eliason, Laurie Reichling, William J. Frazee, Robert A. Horton, Steve Riddle and Yi Gao
Invitrogen Discovery Sciences • 501 Charmany Drive • Madison, Wisconsin 53719 • USA
0.75
0
10 20 30 40 50 60 70 80 90 100
% Conversion (ATPÎADP)
A. Adapta
5µl,
30 min.
®
EC50 = 12 ng/mL
0.001
0.01
0.1
®
1
10
100
450,000
400,000
350,000
300,000
250,000
200,000
150,000
100,000
50,000
0
-50,000
% conversion
ATP to ADP
0
5
10
20
40
60
80
100
®
®
0.1
1
High TR-FRET
The AdaptaTM assay is composed of two steps. First, a standard kinase reaction is performed in the presence or
absence of inhibitor. In the second step, formation of ADP is detected by adding a europium-labeled anti-ADP
antibody, Alexa Fluor® 647 labeled ADP, and EDTA. ADP formed by uninhibited kinase disrupts antibody-tracer
interaction, resulting in a low TR-FRET signal. Inhibited kinase forms less ADP, resulting in an intact antibody-tracer
interaction and a high TR-FRET signal.
Figure 2 – AdaptaTM Assay Sensitivity Compared to Kinase-Glo® Plus Assay
A. AdaptaTM Assay
>4 fold window!
0.75
0.50
0.25
2.0×10
<1.3 fold window!
1.5×10 6
1.0×10 6
5.0×10 5
0
10
20
30
40
50
60
70
80
90 100
% conversion ATP → ADP
0
10
20
30
40
50
60
70
80
90 100
% conversion ATP → ADP
The sensitivity of the AdaptaTM ADP detection assay was compared to the Kinase-Glo® Plus luminescent kinase assay
from Promega Corporation that measures ATP depletion. In the absence of kinase, varying ratios of ATP and ADP
that equaled a total nucleotide concentration of 100 µM were assayed using the standard protocols for each assay
technology. Graph A demonstrates that at 20% conversion of 100 µM ATP, the AdaptaTM assay has a window of >4fold, while the Kinase-Glo® Plus assay graph (B) indicates a very small window of less than 1.3 fold at 20% conversion
of 100 µM ATP. When running a kinase assay, it is desirable to be at < 20% substrate conversion in order to be within
the linear range of the kinase assay.
©2008 Invitrogen Corporation
Adapta™ is a trademark of Invitrogen Corporation
Kinase-Glo® is a registered trademark of Promega Corporation
Fold
Change
N/A
1.05
1.10
1.31
1.84
2.78
5.74
453.19
~ ng/mL
IKKε
N/A
20
35
75
150
250
475
N/A
0.8
0.6
IC50 (nM)
staurosporine, 0.085
0.4
IKK inhibitor IX, 24
0.2
0.1
1
10
100
[compound] (nM)
1000 10000
1000
10000
100000
IC50
PIK3CA/PIK3R1 (p110α /p85α ) 6.3 nM
90 nM
1.4 uM
PIK3C3 (hVPS34)
PIK3CG (p110γ)
50
25
(Lit Ref.)*
8 nM
150 nM
2.3 uM
*Knight et al. Cell 2006, 125 (4), pp. 733-747
250,000
staurosporine, 0.75
0.1
1
10
100
1000
10000
•
The AdaptaTM assay format is a suitable assay choice for any enzyme
reaction that produces ADP. This makes it an excellent choice for the
interrogation of difficult kinase targets such as lipid kinases. We
demonstrated this application with examples of kinase titrations and
inhibitor IC50 determinations for Class I and Class III PI3 kinases.
•
Invitrogen offers a wide selection of lipid kinase products, including purified
lipid kinases, optimized lipid substrates and the AdaptaTM universal kinase
assay kit. For more information, visit www.invitrogen.com/adapta.
1000 10000
[compound] (nM)
Similar IC50 values and rank order potencies were obtained for IKK Inhibitor VII and IX. However, because
the AdaptaTM assay requires 10 fold less kinase per well, a better indication of the potency of staurosporine
is revealed in the Adapta™ assay (85 pM vs 750 pM), which in this case is limited by the amount of kinase
present. Kinase assays were performed using a dose response of the inhibitor, the EC80 concentration of
IKKε, (475 ng/mL for Kinase-Glo® Plus luminescent kinase assay and 40 ng/mL for AdaptaTM assay), 10 µM
ATP and 200 µM IKKtide.
100
Because the AdaptaTM assay uses 10 times less kinase than Kinase-Glo®
Plus Luminescent Kinase assay, tight binding inhibitors can be better
resolved from weaker inhibitors.
IKK inhibitor VII, 35
0.0001 0.001 0.01
10
•
IKK inhibitor IX, 13
50,000
1
The AdaptaTM assay for IKKε requires ten times less kinase than the KinaseGlo® Plus assay and allows inhibitor dose response data to be obtained at
less than 20% substrate conversion while Kinase-Glo® Plus assay requires
closer to 80% conversion.
150,000
IC50 (nM)
0.1
•
200,000
100,000
0.01
Conclusions
• Invitrogen’s AdaptaTM universal kinase assay format detects the formation
of ADP and is much more sensitive than Promega’s Kinase-Glo® Plus ATP
depletion assay. This increased sensitivity results in Z’ values >0.5 at very
low substrate conversion; 5% conversion compared to almost 80% for
Kinase-Glo® Plus Luminescent Kinase Assay for the IKK ε assay.
0
0.0001 0.001 0.01
100
The AdaptaTM universal kinase assay enables the interrogation of difficult kinase targets
such as lipid kinases. Titrations of lipid kinases were performed with 10 µM ATP and 50 µM
PIP2:PS for PIK3CA/PIK3R1, PIK3CG, and PIK3CB/PIK3R1 and 100 µM PI:PS for PIK3C3 .
The EC80 concentrations for each lipid kinase (40 ng/mL for PIK3CA/PIK3R1, 2000 ng/mL for
PIK3Cg, and 150 ng/mL for PIK3C3) were then used to determine the dose response of the
inhibitor PI-103 using the same concentration of ATP and lipid substrate. The resulting IC50
values were in close agreement with literature values.
300,000
IKK inhibitor VII, 48
0
0.00
~ mean
RLU
334,457
317,563
303,110
255,592
181,745
120,297
58,261
738
B. Kinase-Glo® Plus Assay
1.0
6
Emission Ratio
(665/615)
Luminescence
(RLU)
Emission Ratio
(665/615)
1.00
~ ng/mL
IKKε
N/A
14
30
62
150
280
450
N/A
10
[PI-103] (nM)
1000 10000 100000
Figure 5 – IC50 Values of IKKε Inhibitors Using AdaptaTM or Kinase-Glo® Plus
Assays
2.5×10 6
1.25
100
IKKε titrations were performed for each assay technology as described in Figure 3. The AdaptaTM assay (A)
provides a much larger assay window at lower % conversions of ATP to ADP than Kinase-Glo® Plus assay
(B). As a result, 10-fold less kinase is required in the Adapta™ assay format relative to that required for the
Kinase-Glo® Plus assay format. The corresponding tables that list the fold change for each concentration of
kinase and the resulting % conversion of ATP to ADP again highlight the larger assay window for the AdaptaTM
assay at low % conversion.
B. Kinase-Glo® Plus Assay Sensitivity
A. AdaptaTM Assay Sensitivity
10
[IKKε ] (ng/mL)
Fold
Change
N/A
1.90
2.88
4.48
6.47
7.71
8.45
8.78
1
75
0.001
0.01
% conversion
ATP to ADP
0
5
10
20
40
60
80
100
Luminescence (RLU)
Inhibited Reaction
mean
Em Ratio
0.887
0.467
0.308
0.198
0.137
0.115
0.105
0.101
PIK3C3 (hVPS34)
0
[IKKε ] (ng/mL)
Low TR-FRET
PIK3CB/PIK3R1 (p110β/p85α )
125
EC50 = 170 ng/mL
®
Uninhibited Reaction
PIK3CG (p110γ)
100
0.001
1000 10000 100000
PIK3CA/PIK3R1 (p110α /p85α)
B. Lipid Kinase Inhibitors
B. Kinase-Glo Plus Assay
Assay
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
[kinase] (ng/mL)
Figure 4 – IKKε Titrations Using AdaptaTM and Kinase-Glo® Plus Assays
TM
A. Lipid Kinase Titrations
% inhibition
Kinase Reaction
Kinase-Glo® Plus
AdaptaTM
Emission Ratio
(665/615)
Add Antibody
and Tracer
0.25
< 0.00
Figure 1 – AdaptaTM Universal Kinase Assay Schematic
10 µl,
1 hour
0.5
0.50
In an example using IKKε, the AdaptaTM universal kinase assay
achieves a Z’ value of >0.5 at 5% conversion of ATP while the
Kinase-Glo® Plus luminescent kinase assay requires nearly 80%
ATP conversion to achieve a similar Z’ value. The Z’ values for
each assay technology were determined from 24 replicates at
each concentration of an IKKε titration with 10 µM ATP and 200
µM IKKtide. Each assay was developed using the protocol
recommended by the manufacturer. The % conversion of ATP to
ADP that corresponded to each kinase concentration is plotted
versus the calculated Z’ value.
Figure 6 –Using AdaptaTM Assays for Lipid Kinases
Emission Ratio
(665/615)
1.00
Luminescence (RLU)
The Adapta™ universal kinase assay* detects ADP formation, the common product of all
kinase reactions. The assay is based upon the binding of an Alexa Fluor® 647 labeled
ADP analog (tracer) to a europium-labeled anti-ADP antibody. ADP produced by a kinase
competes with the ADP-based tracer for binding to the antibody, resulting in a loss of
FRET, whereas inhibition of kinase activity results in high FRET. The europium:Alexa
Fluor® 647 FRET pair provides a robust signal that is resistant to compound interference,
and utilizes common filter sets such as those used for other TR-FRET technologies. In
contrast to luciferase-coupled ATP depletion assays, AdaptaTM ADP detection assays are
sensitive to very low levels of ADP formation, commonly requiring conversion of less than
10% of ATP to ADP to produce a large signal change. Consequently, much less kinase is
required relative to ATP-depletion based assays that require consumption of > 50% of the
ATP to produce a significant signal change.
Figure 3 – Comparison of Z´ Values from AdaptaTM and Kinase-Glo® Plus
Assays
Z' (n=24)
Introduction
*Adapta™ assays utilize Transcreener™ HTS Assay Platform technology (patent pending) under license from
Bellbrook Labs, LLC.