Product Pathways - HTScan Kinase Assay Kits
HTScan® FLT3 Kinase Assay Kit #7743
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-Tyrosine Mouse mAb (P-Tyr-100) # 9411 | 30 microliters |
| HTScan® Tyrosine Kinase Buffer (4X) # 9805 | 15 milliliters |
| DTT (1000X, 1.25 M) | 80 microliters |
| Gastrin Precursor (Tyr87) Biotinylated Peptide # 1310 | 1.25 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| FLT3 Kinase # 7357 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human FLT3 kinase. It includes active FLT3 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine antibody for detection of the phosphorylated form of the substrate peptide
Molecular Weights
Peptide substrate, Biotin-peptide: 2,853 Daltons. GST-FLT3 Kinase: 82 kDa.
Peptide Core Sequence
EAY*GW
Kinase Assay - Radiometric
Figure 1. FLT3 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: MBP 200 ng/μL, and variable amounts of FLT3.
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of FLT3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of FLT3 substrate peptide (#1310) by FLT3 kinase. In a 50 µl reaction, 50 ng FLT3, 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 3. Dose dependence curve of FLT3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by FLT3 kinase. In a 50 µl reaction, increasing amounts of FLT3 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 4. Peptide concentration dependence of FLT3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by FLT3 kinase. In a 50 µl reaction, 50 ng of FLT3 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 FLT3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of FLT3 substrate peptide (#1310) by FLT3 kinase. In a 50 µl reaction, 50 ng FLT3 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 FLT3 (Arg571-Ser993) (GenBank accession No. NM_004119) 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 Tyrosine Kinase Substrate Screening Kit #7450. Phospho-Tyrosine mAb (P-Tyr-100) #9411 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified FLT3 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the FLT3 kinas 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 FLT3 activity using the FLT3 substrate peptide provided in this kit. FLT3 sensitivity to the inhibitor staurosporine was measured using the FLT3 substrate peptide provided in this kit [Fig.5].
Background
FMS-related tyrosine kinase 3 (FLT3, also called Flk2), is a member of the type III receptor tyrosine kinase family, which includes c-Kit, PDGFR and M-CSF receptors. FLT3 is expressed on early hematopoietic progenitor cells and supports growth and differentiation within the hematopoietic system (1,2). FLT3 is activated after binding with its ligand FL, which results in a cascade of tyrosine autophosphorylation and tyrosine phosphorylation of downstream targets (3). The p85 subunit of PI3 kinase, SHP2, GRB2 and Shc are associated with FLT3 after FL stimulation (4-6). Tyr589/591 is located in the juxtamembrane region of FLT3 and may play an important role in regulation of FLT3 tyrosine kinase activity. Somatic mutations of FLT3 consisting of internal tandem duplications (ITDs) occur in 20% of patients with acute myeloid leukemia (7).
- Shurin, M.R. et al. (1998) Cytokine Growth Factor Rev. 9, 37-48.
- Naoe, T. et al. (2001) Cancer Chemother. Pharmacol. 48 Suppl1, S27-S30.
- Namikawa, R. et al. (1996) Stem Cells 14, 388-395.
- Beslu, N. et al. (1996) J. Biol. Chem. 271, 20075-20081.
- Zhang, S. and Broxmeyer, H.E. (2000) Biochem. Biophys. Res. Commun. 277, 195-199.
- Zhang, S. et al. (1999) J. Leukoc. Biol. 65, 372-380.
- Mizuki, M. et al. (2000) Blood 96, 3907-3914.
Application References
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