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7412
HTScan® PKD/PKCμ Kinase Assay Kit
Cellular Assay Kits
Assay Kit

HTScan® PKD/PKCμ Kinase Assay Kit #7412

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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.)
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.)
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.
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.)
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.)
Inquiry Info.# 7412

Product 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 Formula Peptide substrate, Biotin-peptide: 2,326 Daltons. GST-PKD/PKCmu Kinase domain: 133 kDa.

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).

Pathways

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For Research Use Only. Not for Use in Diagnostic Procedures.
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