Development and Standardization

of a non-radioactive Cdk5 specific

kinase assay

Development of a non-radioactive Cdk5-ADPglo-kinase

assay for high throughput Cdk5 activity screening.
We
have developed a nonradioactive Cdk5 specific kinase assay (Cdk5-ADP gloKinase) in our lab using a Cdk5 specific substrate (NF-H peptide or histone) and
ADPglo system (1). This assay provides a universal, homogeneous, highthroughput screening method to measure Cdk5 kinase activity by quantifying the
amount of ADP produced during kinase reaction. The Cdk5-ADP glo-Kinase assay
is performed in a multiwall plate and can detect kinase activity in a reaction
volume as low as 5l. The principal of assay is described in the figure. In brief,
first kinase reaction is performed using Cdk5 kinase, NF-H peptide substrate
and cold ATP in a kinase reaction buffer. Second, equal volume of ADP-Glo
reagent is added to terminate the kinase reaction and deplete the remaining
ATP. Third, the Kinase detection reagent is added to simultaneously convert
ADP to ATP and allow the newly synthesized ATP to be measured using a
luciferase/luciferin reaction. Fourth, the light generated is measured using a
luminometer. Luminescence is correlated to ADP concentrations by using an
ATP-to-ADP conversion curve (2). This assay is sensitive enough to detect very
low amounts of ADP (20nM) and can detect generated ADP in a reaction
containing up to 1mM ATP in a linear fashion. The luminescent signal generated
is proportional to the ADP concentration produced and is correlated with kinase
activity (3). Because of its non-radioactive nature and high sensitivity this assay
is ideal for high-throughput screening of Cdk5 kinase activity in multiple samples
because a small amount of enzyme and low ATP-to-ADP conversion generate a
high signal-to background ratio.
1)

Hu Li, Rachel D. Totoritis, Leng A. Lor, Benjamin Schwartz, Peter Caprioli, Anthony J.
Jurewicz and Guofeng Zhang. Evaluation of an Antibody-Free ADP Detection Assay: ADPGlo .Assay Drug Dev Technol. 2009 Dec, 7(6): 598-605.

2)

Zegzouti H, Zdanovskaia M, Hsiao K, Goueli SA. ADP-Glo: A Bioluminescent and

homogeneous ADP monitoring assay for kinases. Assay Drug Dev Technol. 2009 Dec;
7(6): 560-72.
Sanghera J, Li R, Yan J. Comparison of the luminescent ADP-Glo assay to a standard
radiometric assay for measurement of protein kinase activity. Assay Drug Dev Technol.
2009 Dec; 7(6): 615-22.

3)

Advantages of the Cdk5 ADPglo Kinase Assay

Broad linear range of ATP concentrations (micromolar to millimolar range): Distinguish between ATP
competitive and noncompetitive inhibitors.
Large dynamic range: High signal-to-background ratios at lower percent conversions of ATP to ADP allow use of
smaller amounts of enzyme.
High sensitivity: Detect 0.2pmol ADP with more than twofold difference over background.
Positive correlation: Assay signal increases linearly with increasing product formation.
Luminescence-based ADP detection: Experience less overall assay interference from chemical compounds.
Batch plate processing: The luminescent signal is highly stable
Homogeneous, robust and non-radioactive assay.

1 mM

15
10
r

Luminescence (RLU) x 105

Luminescence (RLU) x 105

20

0.9958

2
3
4
5
% ATP-ADP conversion

10 M

20

0.9920

10 20 30 40 50 60 70 80 90
% ATP-ADP conversion

100 M

15
10
r

Luminescence (RLU) x 103

Luminescence (RLU) x 105

Sensitivity and linearity of the ADPglo Kinase Assay

35

0.9897

10 20 30 40 50 60 70 80 90
% ATP-ADP conversion
1 M

30
25
20
15
10

0.9940

5
0

10 20 30 40 50 60 70 80 90
% ATP-ADP conversion

10 M-ATP
+
15 5 g Histone
10
r

Luminescence (RLU) x 105

Luminescence (RLU) x 105

20

20
15

10

20
30
40
Cdk5/p25 (ng)

-Rosco.

0.9952

50

60

+Rosco.(10 M)

10 M-ATP + 5 g Histone

10
5
0

50.00 25.00 12.50 6.25 3.21

Cdk5/p25 (ng)

1.56

Luminescence (RLU) x 105

Luminescence (RLU) x 105

Specificity and linearity of the Cdk5 ADPglo Kinase Assay

20

10 M-ATP
+
15 20M NF-H peptide
10
r

5
0

20
15

10

20
30
40
Cdk5/p25 (ng)

0.9776

50

60

-Rosco.
+ Rosco. (10 M)
10 M-ATP + 5 g Histone

10
5

50.00 25.00 12.50 6.25 3.21

Cdk5/p25 (ng)

1.56