Product Pathways - HTScan Kinase Assay Kits
HTScan® Pim-1 Kinase Assay Kit #7573
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-Bad (Ser112) Antibody # 9291 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| Pim-1 Kinase # 7572 | 5 micrograms |
| Bad (Ser112) Biotinylated Peptide # 1342 | 1.25 milliliters |
Description
The kit provides a means of performing kinase activity assays with recombinant human Pim-1 kinase. It includes active Pim-1 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-Bad (Ser112): 1,700 Daltons. GST-Pim-1 Kinase: 65 kDa.
Peptide Core Sequence
RS*RHS*S*Y
Kinase Assay - Radiometric
Figure 1. Pim-1 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: Histone H2B 5 µg/50 µl, recombinant Pim-1: 200 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Pim-1 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of substrate peptide (#1342) by Pim-1 kinase. In a 50 µl reaction, increasing amounts of Pim-1 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of Pim-1 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of Pim-1 substrate peptide (#1342) by Pim-1 kinase. In a 50 µl reaction, 50 ng Pim-1, 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.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence of Pim-1 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of substrate peptide (#1342) by Pim-1 kinase. In a 50 µl reaction, 50 ng of Pim-1 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 2. Time course of Pim-1 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291 to detect phosphorylation of Pim-1 substrate peptide (#1342) by Pim-1 kinase. In a 50 µl reaction, 50 ng Pim-1 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 Pim-1 (Met1-Lys313) (GenBank Accession No. NM_002648) 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-Bad (Ser112) Antibody #9291 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified Pim-1 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the Pim-1 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 Pim-1 activity using the Pim-1 substrate peptide provided in this kit. Pim-1 sensitivity to the inhibitor staurosporine was measured using the Pim-1 substrate peptide provided in this kit [Fig.5].
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 is correlated 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).
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- Aho, T.L. et al. (2004) FEBS Lett 571, 43-9.
- Yan, B. et al. (2003) J Biol Chem 278, 45358-67.
Application References
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