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7412
HTScan® PKD/PKCμ Kinase Assay Kit

HTScan® PKD/PKCμ Kinase Assay Kit #7412

This product is discontinued

Kinase Assay - Radiometric Image 1

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.

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Kinase Assay - DELFIA Image 2

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.)

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Kinase Assay - DELFIA Image 3

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.)

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Kinase Assay - DELFIA Image 4

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.)

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Kinase Assay - DELFIA Image 5

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.)

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Product Includes Quantity (with Count) Solution Color
Kinase Buffer (10X) 9802 1 x 15 ml
ATP (10 mM) 9804 1 x 1 ml

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.

Peptide Core Sequence:

RRPS*YRK

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).

  1. Nishizuka, Y. (1984) Nature 308, 693-8.
  2. Keranen, L.M. et al. (1995) Curr Biol 5, 1394-403.
  3. Valverde, A.M. et al. (1994) Proc. Natl. Acad. Sci. 91, 8572-8576.
  4. Johannes, F.J. et al. (1994) J. Biol. Chem. 269, 6140-6148.
  5. Iglesias, T. et al. (1998) J. Biol. Chem. 273, 27662-27667.
  6. Matthews, S.A. et al. (1999) J. Biol. Chem. 274, 26543-26549.
Entrez-Gene Id
5587
Swiss-Prot Acc.
Q15139
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.

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