Revision 1

#7576Store at -80C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#Q9P1W9

Entrez-Gene Id:

11040

Product Information

Storage

Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO. Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 100 mM NaCl, 5 mM DTT, 15 mM reduced glutathione and 20% glycerol. Store at –80°C.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full length human Pim-2 (Met1-Pro311) (GenBank Accession No. XM_010208) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Product Description

The kit provides a means of performing kinase activity assays with recombinant human Pim-2 kinase. It includes active Pim-2 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: 1,700 Daltons. GST-Pim-2 Kinase : 64 kDa.

Background

Pim proteins (Pim-1, Pim-2 and Pim-3) are oncogene-encoded serine/threonine kinases (1). Pim-1, a serine/threonine kinase highly expressed in hematopoietic cells, plays a critical role in the transduction of mitogenic signals and is rapidly induced by a variety of growth factors and cytokines (1-4). Pim-1 cooperates with c-Myc in lymphoid cell transformation and protects cells from growth factor withdrawal and genotoxic stress-induced apoptosis (5,6). Pim-1 also enhances the transcriptional activity of c-Myb through direct phosphorylation within the c-Myb DNA binding domain as well as phosphorylation of the transcriptional coactivator p100 (7,8). Hypermutations of the Pim-1 gene are found in B-cell diffuse large cell lymphomas (9). Phosphorylation of Pim-1 at Tyr218 by Etk occurs following IL-6 stimulation and correlates with an increase in Pim-1 activity (10). Various Pim substrates have been identified; Bad is phosphorylated by both Pim-1 and Pim-2 at Ser112 and this phosphorylation reverses Bad-induced cell apoptosis (11,12).

  1. Mikkers, H. et al. (2004) Mol Cell Biol 24, 6104-15.
  2. Selten, G. et al. (1986) Cell 46, 603-11.
  3. Meeker, T.C. et al. (1987) J Cell Biochem 35, 105-12.
  4. Dautry, F. et al. (1988) J Biol Chem 263, 17615-20.
  5. Möröy, T. et al. (1993) Proc Natl Acad Sci USA 90, 10734-8.
  6. Lilly, M. and Kraft, A. (1997) Cancer Res 57, 5348-55.
  7. Leverson, J.D. et al. (1998) Mol Cell 2, 417-25.
  8. Winn, L.M. et al. (2003) Cell Cycle 2, 258-62.
  9. Pasqualucci, L. et al. (2001) Nature 412, 341-6.
  10. Kim, O. et al. (2004) Oncogene 23, 1838-44.
  11. Aho, T.L. et al. (2004) FEBS Lett 571, 43-9.
  12. Yan, B. et al. (2003) J Biol Chem 278, 45358-67.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
#7576

HTScan® Pim-2 Kinase Assay Kit

HTScan® Pim-2 Kinase Assay Kit: Image 1 Expand Image
Figure 3. Dose dependence curve of Pim-2 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of substrate peptide (#1342) by Pim-2 kinase. In a 50 µl reaction, increasing amounts of Pim-2 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® Pim-2 Kinase Assay Kit: Image 2 Expand Image
Figure 5. Staurosporine inhibition of Pim-2 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of Pim-2 substrate peptide (#1342) by Pim-2 kinase. In a 50 µl reaction, 50 ng Pim-2, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® Pim-2 Kinase Assay Kit: Image 3 Expand Image
Figure 4. Peptide concentration dependence of Pim-2 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of substrate peptide (#1342) by Pim-2 kinase. In a 50 µl reaction, 50 ng of Pim-2 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® Pim-2 Kinase Assay Kit: Image 4 Expand Image
Figure 2. Time course of Pim-2 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of Pim-2 substrate peptide (#1342) by Pim-2 kinase. In a 50 µl reaction, 50 ng Pim-2 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® Pim-2 Kinase Assay Kit: Image 5 Expand Image
Figure 1. Pim-2 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, ATP (variable), 2.5 µg/50 µl PEG20,000, Substrate: R11-S6-peptide5 µg/50 µl, recombinant Pim-2: 200 ng/50 µl.
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.