c-Src Kinase Inhibitor Screening Assay Kit

Intended Use

The c-Src Kinase Inhibitor Screening Assay Kit is designed to measure the activities of recombinant catalytic domain of c-Src for the rapid and sensitive evaluation of inhibitors or activators. The phosphotyrosine specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phosphotyrosine residue in the recombinant "Tyrosine kinase-substrate-1", which is efficiently phosphorylated by c-Src in vitro.

Applications of this kit include:
1) Screening inhibitors or activators of recombinant catalytic domain of c-Src.
2) Detecting the effects of pharmacological agents on recombinant catalytic domain of c-Src.

This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.

Storage

Upon receipt store all components at 4°C.
Don't expose reagents to excessive light.

Performance Characteristics

The c-Src Kinase Inhibitor Screening Assay Kit has been shown to detect the kinase activity of recombinant catalytic domain of c-Src. The assay shows good linearity of sample response.

General Description

The Src family of non-receptor protein tyrosine kinases plays critical roles in a variety of cellular signal transduction pathways, regulating such diverse processes as cell division, motility, adhesion, angiogenesis, and survival. Constitutively activated variants of Src family kinases, including the viral oncoproteins v-Src and v-Yes, are capable of inducing malignant transformation of a variety of cell types. Src family kinases, most notably although not exclusively Src, are frequently overexpressed and/or aberrantly activated in a variety of epithelial and non-epithelial cancers. Activation is very common in colorectal and breast cancers, and somewhat less frequent in melanomas, ovarian cancer, gastric cancer, head and neck cancers, pancreatic cancer, lung cancer, brain cancers, and blood cancers. Further, the extent of increased Src family activity often correlates with malignant potential and patient survival. Activation of Src family kinases in human cancers may occur through a variety of mechanisms and is frequently a critical event in tumor progression. Exactly how Src family kinases contribute to individual tumors remains to be defined completely, however they appear to be important for multiple aspects of tumor progression, including proliferation, disruption of cell/cell contacts, migration, invasiveness, resistance to apoptosis, and angiogenesis.
Elevated Src tyrosine kinase activity has been found in colon cancers, particularly in those metastatic to the liver. Studies of the mechanism of Src regulation suggested that Src kinase activity is downregulated by phosphorylation of a critical C-terminal tyrosine (Tyr530 in human Src) and have implied the existence of activating mutations in this C-terminal regulatory region. Irby et al. reported the identification of a truncating mutation in Src at codon 531 in 12% of cases of advanced human colon cancer tested and demonstrated that the mutation is activating, transforming, tumorigenic, and metastasis-promoting. The results provided, for the first time, genetic evidence that activating Src mutations may have a role in the malignant progression of human colon cancer.

Measurement of c-Src Kinase activity

The protocol generally regarded as most sensitive for the quantitative measurement of c-Src kinase activity involves incubation of the c-Src kinase sample with substrate, either a natural or synthetic polypeptide (such as poly[Glu,Tyr]4:1), in the presence of Mg2+, Mn2+ and 32P-labeled ATP. The reaction is terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid to precipitate the radiolabeled product. The filter papers are then washed extensively to remove unincorporated radiolabel and the radioactivity is counted. While sensitive, this method is labor-intensive, generates hazardous radioactive waste, and depends on a radioisotope of short half-life. It is particularly unsuitable when kinase assays are only performed on an infrequent basis. The c-Src Kinase Inhibitor Screening Assay Kit uses a horseradish peroxidase coupled anti-phosphotyrosine monoclonal antibody as a reporter molecule in a 96-wells ELISA format. This assay provides a non-isotopic, sensitive and specific method to detect kinase activity of recombinant c-Src catalytic domain.