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 IC 50 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 |
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All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Biochemistry
- Clinical Biochemistry
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Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay. / Nguyen, Binh Thanh; Park, Min; Pyun, Jae Chul; Yoo, Young Sook; Kang, Min Jung.
In: Electrophoresis, Vol. 37, No. 23-24, 01.12.2016, p. 3146-3153.Research output: Contribution to journal › Article
TY - JOUR
T1 - Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay
AU - Nguyen, Binh Thanh
AU - Park, Min
AU - Pyun, Jae Chul
AU - Yoo, Young Sook
AU - Kang, Min Jung
PY - 2016/12/1
Y1 - 2016/12/1
N2 - 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 IC 50 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.
AB - 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 IC 50 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.
UR - http://www.scopus.com/inward/record.url?scp=84996848911&partnerID=8YFLogxK
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U2 - 10.1002/elps.201600330
DO - 10.1002/elps.201600330
M3 - Article
VL - 37
SP - 3146
EP - 3153
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 23-24
ER -