Product Pathways - Tyrosine Kinase/ Adaptors
Src Kinase #7775
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.
Description
Purified recombinant full length human Src 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 Src (Met1-Leu536) (GenBank Accession No. NM_005417) 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-Src fusion protein was analyzed using SDS/PAGE followed by anti-Src Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. 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 and 20 ng/50 µl Recombinant Src.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Src kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411to 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.)
Quality Control
The theoretical molecular weight of the GST-Src fusion protein is 90 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. Src kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Src activity using HTScan™ Src Kinase Assay Kit #7773 [Fig.3].
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
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Companion Products
- 7776 HTScan® Src Kinase Assay Kit
- 1366 Signal Transduction Protein (Tyr160) Biotinylated Peptide
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
- 7450 Tyrosine Kinase Substrate Screening Kit
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.
