Product Pathways - HTScan Kinase Assay Kits
HTScan® Src Kinase Assay Kit #7776
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 |
| ATP (10 mM) # 9804 | 1 milliliters |
| Src Kinase # 7775 | 5 micrograms |
| Signal Transduction Protein (Tyr160) Biotinylated Peptide # 1366 | 1.25 milliliters |
Description
The kit provides a means of performing kinase activity assays with recombinant human Src kinase. It includes active Src 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: 1830 Daltons. GST-Src Kinase: 90 kDa.
Peptide Core Sequence
GIY*DV
Kinase Assay - Radiometric
Figure 1. Src 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: PolyEY, 1 µg/50 µl, recombinant Src: 20 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Src kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1366) by Src kinase. In a 50 µl reaction, increasing amounts of Src 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 Src kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of Src substrate peptide (#1366) by Src kinase. In a 50 µl reaction, 50 ng Src, 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 Src kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1366) by Src kinase. In a 50 µl reaction, 50 ng of Src 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 Src kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of Src substrate peptide (#1366) by Src kinase. In a 50 µl reaction, 50 ng Src 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 Src (Met1-Leu536) 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 Mouse 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 Src kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the Src 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 Src activity using the Src substrate peptide provided in this kit. Src sensitivity to the inhibitor staurosporine was measured using the Src substrate peptide provided in this kit [Fig.5].
Background
The Src family of protein tyrosine kinases (including Src, Lyn, Fyn, Yes, Lck, Blk and Hck) are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. Phosphorylation of Tyr416 in the activation loop of the kinase domain by Csk upregulates enzyme activity, whereas phosphorylation of Tyr527 in the carboxy-terminal tail renders the enzyme less active (2).
- Thomas, S.M. and Brugge, J.S. (1997) Annu. Rev. Cell Dev. Biol. 13, 513-609.
- Hunter, T. (1987) Cell 49, 1-4.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Companion Products
- 7450 Tyrosine Kinase Substrate Screening Kit
- 7775 Src Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1366 Signal Transduction Protein (Tyr160) Biotinylated Peptide
- 9953 Staurosporine
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
