Product Pathways - HTScan Kinase Assay Kits
HTScan® Aurora B Kinase Assay Kit #7513
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-PLK (Ser137) Antibody # 5070 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| PLK (Ser137) Biotinylated Peptide # 1300 | 1.25 milliliters |
| Aurora B Kinase | 2 x 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human Aurora B kinase. It includes active Aurora B kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Biotin-PLK (Ser137): 1,945 Daltons. GST-Aurora B Kinase: 70 kDa.
Peptide Core Sequence
RRS*LL
Kinase Assay - Radiometric
Figure 1. Aurora B 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: Tetra (LRRLSLG), 5 µg/50 µl, recombinant Aurora B: 200 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Aurora B kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by Aurora B kinase. In a 50 µl reaction, increasing amounts of Aurora B 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 Aurora B kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of Aurora B substrate peptide (#1300) by Aurora B kinase. In a 50 µl reaction, 100 ng Aurora B, 1.5 µM substrate peptide, 50 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 Aurora B kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by Aurora B kinase. In a 50 µl reaction, 100 ng of Aurora B 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 Aurora B kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of Aurora B substrate peptide (#1300) by Aurora B kinase. In a 50 µl reaction, 100 ng Aurora B 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 a fragment of human Aurora B (Ala2-Ala344) (GenBank Accession No. NM_004217) 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-PLK (Ser137) Antibody #5070 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified Aurora B kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the Aurora B kinase 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 Aurora B activity using the Aurora B substrate peptide provided in this kit. Aurora B sensitivity to the inhibitor staurosporine was measured using the Aurora B substrate peptide provided in this kit [Fig.5].
Background
Aurora kinases belong to a highly conserved family of mitotic serine/threonine kinases with three members identified among mammals: Aurora A, Aurora B and Aurora C (1,2). Studies on the temporal expression pattern and subcellular localization of Aurora kinases in mitotic cells suggest an association with mitotic structure. Their functional influences span from G2 to cytokinesis and may be involved in key cell cycle events such as centrosome duplication, chromosome bi-orientation and segregation, cleavage furrow positioning and ingression (3). Aurora A is detected at the centrosomes, along mitotic spindle microtubules and in the cytoplasm of mitotically proliferating cells. Aurora A protein levels are low during G1 and S phases and peak during the G2/M phase of the cell cycle. Phosphorylation of Aurora A at Thr288 in its catalytic domain increases kinase activity. Aurora A is involved in centrosome separation, maturation and spindle assembly and stability. Expression of Aurora B protein also peaks during the G2/M phase of the cell cycle, while kinase activity peaks at the transition from metaphase to the end of mitosis. Aurora B associates with chromosomes during prophase prior to relocalizing to the spindle at anaphase. Aurora B regulates chromosome segregation through the control of microtubule-kinetochore attachment and cytokinesis. Expression of both Aurora A and Aurora B during the G2/M phase transition is tightly coordinated with histone H3 phosphorylation (4,5), while overexpression of both kinases is seen in a variety of human cancers (2,4). Aurora C localizes to the centrosome from anaphase to cytokinesis and both mRNA and protein levels peak during G2/M phase. Although typical Aurora C expression is limited to the testis, overexpression of Aurora C is detected in various cancer cell lines (6).
- Warner, S.L. et al. (2003) Mol. Cancer Ther. 2, 589-595.
- Katayama , H. et al. (2003) Cancer Metastasis Rev. 22, 451-464.
- Andrews, P.D. et al. (2003) Curr. Opin. Cell Biol. 15, 672-683.
- Pascreau, G. et al. (2003) Prog. Cell Cycle Res. 5, 369-374.
- Crosio, C. et al. (2002) Mol. Cell. Biol. 22, 874-885.
- Kimura, M. et al. (1999) J. Biol. Chem. 274, 7334-7340.
Application References
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