Product Pathways - HTScan Kinase Assay Kits
HTScan® PKD/PKCμ Kinase Assay Kit #7412
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
| Kit Includes | Quantity |
|---|---|
| Phospho-PKA Substrate (RRXS/T) (100G7E) Rabbit mAb # 9624 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| CREB (Ser133) Biotinylated Peptide # 1331 | 1.25 milliliters |
| PKCμ Kinase # 7602 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human PKD/PKCmu kinase. It includes active PKD/PKCmu kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Peptide substrate, Biotin-peptide: 2,326 Daltons. GST-PKD/PKCmu Kinase domain: 133 kDa.
Peptide Core Sequence
RRPS*YRK
Kinase Assay - Radiometric
Figure 1. PKD/PKCmu kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 1.32 mM CaCl2, 1 mM EDTA, 1.25 mM EGTA, 0.25 μg/50 μl phosphatidylserine, 50 ng/50 μl 1.2 Dioleyl-glycerol, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: tetra (LRRWSLG), 0.5 µg/50 µl and recombinant PKD/PKCmu: 200 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of PKD/PKCmu kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKD/PKCmu kinase. In a 50 µl reaction, increasing amounts of PKD/PKCmu and 1.5 µM substrate peptide were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of PKD/PKCmu kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKD/PKCmu substrate peptide (#1331) by PKD/PKCmu kinase. In a 50 µl reaction, 10 ng PKD/PKCmu, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence of PKD/PKCmu kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKD/PKCmu kinase. In a 50 µl reaction, 10 ng of PKD/PKCmu and increasing concentrations of substrate peptide were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 2. Time course of PKD/PKCmu kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKD/PKCmu substrate peptide (#1331) by PKD/PKCmu kinase. In a 50 µl reaction, 10 ng PKD/PKCmu and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full length human PKD/PKCmu (Met1-Leu912) (GenBank Accession No. NM_002742) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Quality Control
The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified PKD/PKCmu kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. PKD/PKCmu kinase specific activity was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify PKD/PKCmu activity using the PKD/PKCmu substrate peptide provided in this kit. PKD/PKCmu sensitivity to the inhibitor staurosporine was measured using the PKD/PKCmu substrate peptide provided in this kit [Fig.5].
Background
Activation of PKC is one of the earliest events in a cascade leading to a variety of cellular responses such as secretion, gene expression, proliferation and muscle contraction (1,2). Protein kinase D (PKD), also called PKCμ, is a serine/threonine kinase whose activation is dependent on the phosphorylation of two activation loop sites, Ser744 and Ser748, via a PKC-dependent signaling pathway (3-5). In addition to the two activation loop sites, the carboxy-terminal Ser916 has been identified as an autophosphorylation site for PKD/PKCμ. Phosphorylation at Ser916 correlates with PKD/PKCμ catalytic activity (6).
- Nishizuka, Y. (1984) Nature 308, 693-698.
- Keranen, L.M. (1995) Curr. Biol. 5, 1394-1403.
- Valverde, A.M. et al. (1994) Proc. Natl. Acad. Sci. 91, 8572-8576.
- Johannes, F.J. et al. (1994) J. Biol. Chem. 269, 6140-6148.
- Iglesias, T. et al. (1998) J. Biol. Chem. 273, 27662-27667.
- Matthews, S.A. et al. (1999) J. Biol. Chem. 274, 26543-26549.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Protocols
Companion Products
- 7602 PKCμ Kinase
- 9624 Phospho-PKA Substrate (RRXS/T) (100G7E) Rabbit mAb
- 1331 CREB (Ser133) Biotinylated Peptide
- 9953 Staurosporine
- 9802 Kinase Buffer (10X)
- 9804 ATP (10 mM)
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
