Binh Thanh Nguyen Park, Min Pyun, Jae-Chul Yoo, Young Sook Kang, Min-Jung 2024-01-20T02:34:22Z 2024-01-20T02:34:22Z 2021-09-05 2016-12 0173-0835 https://pubs.kist.re.kr/handle/201004/123357 An assay for protein kinase C delta (PKC delta) 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 PKC delta was studied in vitro using the human gastric cancer cell line MKN-1. The use of PKC delta inhibitor candidates, including G.6983, bisindolylmaleimide II, staurosporine, and rottlerin in the assay resulted in IC50 values of 50 nM, 15 nM, 795 nM, and 4 mu M, 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. English John Wiley & Sons Ltd. Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay Article 10.1002/elps.201600330 1 Electrophoresis, v.37, no.23-24, pp.3146 - 3153 Electrophoresis 37 23-24 3146 3153 N scie scopus 000392421400014 2-s2.0-84996848911 Biochemical Research Methods Chemistry, Analytical Biochemistry & Molecular Biology Chemistry Article PROTEIN-KINASE-C INDUCED FLUORESCENCE DETECTION CAPILLARY-ELECTROPHORESIS EXPRESSION DELTA PATHWAYS INSIGHTS CANCER CELLS CE-LIF Kinase Protein Kinase C Screening of kinase inhibitor