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7579
HTScan® PKCα Kinase Assay Kit

HTScan® PKCα Kinase Assay Kit #7579

This product is discontinued

Kinase Assay - Radiometric Image 1

Figure 1. PKCalpha kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 1 µM ATP, 2.5 µg/50 µl PEG20,000, Substrate: Histone H1, 5 µg/50 µl and Recombinant PKCalpha: 25 ng/50 µl.

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Kinase Assay - DELFIA Image 2

Figure 3. Dose dependence curve of PKCalpha kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCalpha kinase. In a 50 µl reaction, increasing amounts of PKCalpha 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 PKCalpha kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCalpha substrate peptide (#1331) by PKCalpha kinase. In a 50 µl reaction, 10 ng PKCalpha, 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 PKCalpha kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCalpha kinase. In a 50 µl reaction, 10 ng of PKCalpha 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 PKCalpha kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCalpha substrate peptide (#1331) by PKCalpha kinase. In a 50 µl reaction, 10 ng PKCalpha and 1.5 uM 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
ATP (10 mM) 9804 1 x 1 ml
Kinase Buffer (10X) 9802 1 x 15 ml

The kit provides a means of performing kinase activity assays with recombinant human PKCalpha kinase. It includes active PKCalpha 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-CREB (Ser133): 2,326 Daltons. GST-PKCalpha Kinase: 110 kDa.

Peptide Core Sequence:

RRPS*YRK

Activation of protein kinase C (PKC) is one of the earliest events in a cascade that controls a variety of cellular responses, including secretion, gene expression, proliferation, and muscle contraction (1,2). PKC isoforms belong to three groups based on calcium dependency and activators. Classical PKCs are calcium-dependent via their C2 domains and are activated by phosphatidylserine (PS), diacylglycerol (DAG), and phorbol esters (TPA, PMA) through their cysteine-rich C1 domains. Both novel and atypical PKCs are calcium-independent, but only novel PKCs are activated by PS, DAG, and phorbol esters (3-5). Members of these three PKC groups contain a pseudo-substrate or autoinhibitory domain that binds to substrate-binding sites in the catalytic domain to prevent activation in the absence of cofactors or activators. Control of PKC activity is regulated through three distinct phosphorylation events. Phosphorylation occurs in vivo at Thr500 in the activation loop, at Thr641 through autophosphorylation, and at the carboxy-terminal hydrophobic site Ser660 (2). Atypical PKC isoforms lack hydrophobic region phosphorylation, which correlates with the presence of glutamic acid rather than the serine or threonine residues found in more typical PKC isoforms. The enzyme PDK1 or a close relative is responsible for PKC activation. A recent addition to the PKC superfamily is PKCμ (PKD), which is regulated by DAG and TPA through its C1 domain. PKD is distinguished by the presence of a PH domain and by its unique substrate recognition and Golgi localization (6). PKC-related kinases (PRK) lack the C1 domain and do not respond to DAG or phorbol esters. Phosphatidylinositol lipids activate PRKs, and small Rho-family GTPases bind to the homology region 1 (HR1) to regulate PRK kinase activity (7).

PKCalpha consists of 672 amino acids and is expressed ubiquitously. It performs important roles in the control of major cellular functions such as cell proliferation, apoptosis, differentiation and motility. PKCalpha is also associated with the malignant phenotype of several tumors, such as gliomas and breast cancers (10,11).

  1. Nishizuka, Y. (1984) Nature 308, 693-8.
  2. Keranen, L.M. et al. (1995) Curr Biol 5, 1394-403.
  3. Mellor, H. and Parker, P.J. (1998) Biochem J 332 ( Pt 2), 281-92.
  4. Ron, D. and Kazanietz, M.G. (1999) FASEB J 13, 1658-76.
  5. Moscat, J. and Diaz-Meco, M.T. (2000) EMBO Rep 1, 399-403.
  6. Baron, C.L. and Malhotra, V. (2002) Science 295, 325-8.
  7. Flynn, P. et al. (2000) J Biol Chem 275, 11064-70.
  8. Nakashima, S. (2002) J. Biochem. (Tokyo) 132, 669-75.
  9. Mackay, H.J. and Twelves, C.J. (2003) Endocr. Relat. Cancer 10, 389-96.
Entrez-Gene Id
5578
Swiss-Prot Acc.
P17252
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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