Promega’s ADP-Glo™ Kinase Assay performed on
BMG LABTECH’s PHERAstar FS and POLARstar Omega
Franka Ganske, BMG LABTECH Germany and EJ Dell, BMG LABTECH, NC, USA
Sarah Schultz, Promega Corp., Madison, WI, USA
ADP-GloTM Kinase Assay is a homogeneous luminescent assay
to detect ADP
ADP concentrations ranging from 1 mM to 0.01 µM can
be detected
Kit was successfully performed on the PHERAstar FS and
POLARstar Omega using on-board injectors
Introduction
Application Note 202, Rev. 12/2009
help of a luciferase/luciferin reaction. The luminescence measured
after incubation is proportional to the ADP concentration generated
during the enzymatic reaction.
Materials and Methods
White 384-well small volume plates from Greiner
ADP-Glo™ Kinase Assay from Promega, including ATP and ADP
PHERAstar FS or POLARstar Omega microplate reader (Fig. 2)
Kinases are a large and diverse group of enzymes that are involved
in many cellular metabolic and regulatory processes. During the
kinase reaction, substrates are phosphorylated while ATP is converted
into ADP. Screening for active kinases or for kinase inhibitors is
an important tool for the development of new drugs. To fulfil this
need, Promega developed a kinase assay that is based on
luminescence and the by-product ADP. The ADP-Glo™ Kinase assay
is a universal, homogeneous, high-throughput screening method to
measure kinase activity by quantifying the amount of ADP produced
during the kinase reaction. This assay can be used to screen for any
ADP-generating enzyme.
In this application note we will show results of the ADP-Glo Kinase
assay obtained with the PHERAstar FS and POLARstar Omega multidetection microplate readers from BMG LABTECH.
Assay Principle
The principle of the assay is presented in figure 1.
+ sample
ATP
Kinase
reaction
ATP + ADP
+ ADP-Glo
Reagent
TM
ATP Depletion Step
Fig. 2: BMG LABTECH’s multidetection microplate readers PHERAstar FS and
POLARstar Omega
To mimic an enzymatic reaction, an ADP/ATP standard curve was
prepared from the nucleotide stock solutions that were supplied
with the kit. The assay allows ADP measurements from 1 mM ADP
to 0.01 µM ADP. Four different ADP/ATP standard curves were prepared: 1 mM, 100 µM, 10 µM and 1 µM. As an example, the dilution
table for a 1 mM ATP/ADP standard dilution is given below. All
other standard curves were diluted accordingly.
mM
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10 S11 S12
ADP
1
0.8
0.6
0.4
0.2
0.1 0.05 0.04 0.03 0.02 0.01
0
ATP
0
0.2
0.4
0.6
0.8
0.9 0.95 0.96 0.97 0.98 0.99
1
+ Kinase Detection
Reagent
ADP Detection Step
ATP
ADP
Incubation
Luminescence
Fig. 1: Principle of the ADP-Glo™ Kinase Assay.
The assay consists of two steps. After the enzymatic reaction is
finished, ATP and ADP are present in the well. The first step is to
add the ADP-Glo™ Reagent resulting in the elimination of the
remaining ATP. The second step is to convert the remaining ADP into
ATP by adding Kinase Detection Reagent. This reagent also contains
everything needed to measure the newly generated ATP with the
Five µL of standard dilution was mixed with 5 µL of ADP-Glo™
reagent followed by a 40 min incubation at room temperature.
After that 10 µL of Kinase Detection Reagent was then injected
with onboard injectors. The signal was measured each minute
for 40 minutes. Alternatively it is possible to measure endpoint
luminescence after a 40-60 min incubation at room temperature.
Instrument settings
POLARstar Omega
Detection Mode:
Measurement time:
Cycle Time:
Cycles:
Optics:
Gain:
for a kinetic reaction on a PHERAstar FS or
Luminescence, plate mode
1 sec
60 sec
40
Luminescence Module (PHERAstar FS)
2 mm optic (POLARstar Omega)
Optimized for the ADP concentration
Results and Discussion
Fig. 3 shows a signal curve taken from the PHERAstar FS for a 1 mM
ADP/ATP standard dilution.
These fits can be seen in figures 4 and 5 for a 1 mM nucleotide
standard curve as well as in figures 6 and 7 for a 1 µM nucleotide
standard curve.
6000
PHERAstar FS
5000
1 mM ADP
8.8E5
8.0E5
4000
0.8 mM ADP
6.4E5
RLU
Luminescence in RLU
7.2E5
3000
R2 = 0.9958
5.6E5
0.6 mM ADP
4.8E5
2000
4.0E5
0.4 mM ADP
3.2E5
1000
2.4E5
1.6E5
0.2 mM ADP
8.0E4
0.1 mM 0 mM ADP
0
0
4
8
12
16
20
24
28
32
0
0
36
1200
Fig. 3: Signal curves for several dilutions of ADP/ATP standards measured
on the PHERAstar FS. The gain was set to 2400.
PHERAstar FS
0.6
0.8
1
1.2
POLARstar Omega
1000
800
RLU
The signal curves show that already after 20 min the signal is very
stable. The 12 standards show different signal heights according
to their ADP concentration. The last 10 data points of the signal
curves were averaged and taken as a base for a linear regression fit.
1.8E6
0.4
µM ADP
Time in minutes
2.0E6
0.2
600
R2 = 0.9937
400
200
1.6E6
0
1.4E6
RLU
0.4
0.6
0.8
1
1.2
µM ADP
1.2E6
R2 = 0.9985
Fig. 6 and 7: One µM ADP/ATP standard curve for PHERAstar FS and
POLARstar Omega.
1.0E6
8.0E5
6.0E5
4.0E5
2.0E5
0
0
100
200
300
400
500
600
700
800
900
µM ADP
5.0E4
POLARstar Omega
4.5E4
The standards curves obtained on the PHERAstar FS and POLARstar
Omega look similar, but note how the FS has a steeper slope
at lower ADP concentration. This reflects the fact that the
PHERAstar FS HTS instrument is more sensitive and has a better
LOD, which is expected. It is possible to measure standard curves
from 1 mM to 1 µM ADP using the same gain settings indicating a
high assay window for both instruments.
Conclusion
4.0E4
The PHERAstar FS as well the POLARstar Omega offer the ability to
perform the ADP-Glo™ Kinase Assay from Promega in a 384-well
format using only 20 µL. Using either instrument, it is possible to
choose to follow the reaction online using the kinetic mode or as
an endpoint measurement.
3.5E4
3.0E4
RLU
0.2
2.5E4
R2 = 0.9989
2.0E4
1.5E4
1.0E4
5.0E3
0
0
200
400
600
800
1000
1200
µM ADP
Fig. 4 and 5: One mM ADP/ATP standard curve for PHERAstar FS and
POLARstar Omega.
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