Product Pathways - Lymphocyte Signaling
Pim-1 Kinase #7572
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Description
Purified recombinant full length human Pim-1 kinase, supplied as a GST fusion protein.
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.
Gel Staining
Figure 1. The purity of the GST-Pim-1 fusion protein was analyzed using SDS/PAGE followed by anti-Pim-1 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. 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 and 200 ng/50 µl Recombinant Pim-1.
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.)
Quality Control
The theoretical molecular weight of the GST-Pim-1 fusion protein is 65 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. Pim-1 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Pim-1 activity using HTScan™ Pim-1 Kinase Assay Kit #7573 [Fig.3].
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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Application References
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