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4.3.3. Quantification of CaMKII activity in a spectrophotometric
assay
The activity of the in silico hits were quantified by CaMKII drug
discovery kit (Cyclex, Japan). The 96-well plate of the kit is pre-
coated with ‘Syntide-2’ phosphorylated by CaMKII. The detector
antibody (MS-6E6) is conjugated to horseradish peroxidase and
specifically detects the phosphorylated form of ‘Syntide-2’.
To perform the assay, the tested hits were first diluted with the
kinase buffer provided with the kit. The hits’ solutions were then
pipetted into assay wells to yield final concentrations of 0.1, 1.0
and 10
CaMKII was added to each well as aqueous solution (0.01 units
in 10 L) and allowed to phosphorylate the bound substrate in
lM and occasionally at 0.2 and 0.02 lM. Subsequently,
l
the presence of CaCl2, Calmodulin and ATP. The amount of phos-
phorylated substrate was measured by allowing it to bind with
the detector antibody conjugated to horseradish peroxidase, which
then catalyzes the conversion of the chromogenic substrate tetra-
methylbenzidine (TMB) from a colorless solution to a blue solution.
The color is quantified by spectrophotometry and reflects the rela-
tive activity of CaMKII. Samples’ absorbances were determined at a
wavelength of 650 nm using a plate reader (Bio-Tek instruments
ELx 800, USA). Inhibition of CaMKII was calculated as percent
activity of the uninhibited CaMKII enzyme control. KN-62 was used
as positive control (Table 6). Negative controls were prepared by
adding the enzyme to the kinase reaction buffer only.40
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Acknowledgments
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This project was partially sponsored by the Faculty of Graduate
Studies (This work is part of Ph.D. Thesis of Rand Omar Shahin).
The authors thank the Deanship of Scientific Research and
Hamdi-Mango Center for Scientific Research at the University of
Jordan for their generous funds.
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Supplementary data
39. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
2001, 46, 3.
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Supplementary data associated with this article can be found, in
41. CycLex, CaM kinase II Assay Kit (Cat# CY-1173) Users’ Manual; CycLex Co., Ltd:
Ina, Nagano, Japan, 2009.
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