Abstract
An assay for protein kinase C delta (PKCδ) activity based on the quantification of a synthetic substrate using capillary electrophoresis with laser-induced fluorescence detection was developed. The peptides labeled with fluorescein isothiocyanate F-ERK (where ERK is extracellular signal-regulated kinase) and the phosphorylated form, P-F-ERK, were utilized for the method development and validation. The migration time of F-ERK and P-F-ERK were 6.3 ± 0.1 and 8.7 ± 0.2 min, respectively. LOD and LOQ values of F-ERK were 2 and 6 ng/mL and those of P-F-ERK were 4 and 12 ng/mL. The correlation coefficients obtained from two standard curves were approximately 0.99. The reproducibility and accuracy of the method for F-ERK ranged 1.5–4.7 and 86–109%, respectively, and those for P-F-ERK were 1.6–6.1 and 93–109%, respectively. The activity of PKCδ was studied in vitro using the human gastric cancer cell line MKN-1. The use of PKCδ inhibitor candidates, including Gӧ6983, bisindolylmaleimide II, staurosporine, and rottlerin in the assay resulted in IC50 values of 50 nM, 15 nM, 795 nM, and 4 μM, respectively. Comparison of our assay with a commercial PKC kit revealed that our assay is more adaptable to differing enzyme isoforms. This method has potential for high throughput screening for kinase inhibitors as part of a drug discovery program.
Original language | English |
---|---|
Pages (from-to) | 3146-3153 |
Number of pages | 8 |
Journal | Electrophoresis |
Volume | 37 |
Issue number | 23-24 |
DOIs | |
Publication status | Published - 2016 Dec 1 |
Bibliographical note
Funding Information:This study was supported by the Creative Fusion Research Program through the Creative Allied Project funded by Korea Research Council of Fundamental Science and Technology (CAP-12-1) and Korea Institute of Science and Technology (KIST) Institutional Program (project nos. 2N41080 and 2E26110).
Publisher Copyright:
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Biochemistry
- Clinical Biochemistry