Key features and details
- Sample type: Adherent cells, Suspension cells
- Detection method: Colorimetric
- Assay type: Cell-based (quantitative)
- Reacts with: Mouse, Human
Product namePhospho-c-Src (Y418) + Total In-Cell ELISA Kit
See all Src kits
Sample typeAdherent cells, Suspension cells
Assay typeCell-based (quantitative)
Assay durationMultiple steps standard assay
Species reactivityReacts with: Mouse, Human
Phospho-c-Src (Y418) + Total In-Cell ELISA Kit (ab207461) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. This kit is designed specifically to quantify activated (phosphorylated) c-Src and/or total c-Src. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated c-Src or total c-Src. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified colorimetric readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.
The Phospho-c-Src (Y418) + Total In-Cell ELISA Kit (ab207461) contains two 96-well plates and two primary antibodies. The phospho-c-Src antibody is specific for phosphorylated c-Src and was raised against a synthetic phospho-peptide corresponding to residues surrounding Tyr418 of human c-Src. This antibody recognizes c-Src when phosphorylated at this site and also recognizes Fyn and c-Yes. The total-c-Src antibody recognizes c-Src proteins regardless of the phosphorylation state. This kit can be used to study phosphorylated c-Src relative to cell number or be used to determine c-Src phosphorylation relative to the total c-Src protein found in the cells. Once the phospho-c-Src and total- c-Src signals have been normalized for cell number, a comparison of the ratio of phosphorylated c-Src to total c-Src for each of the cell growth conditions can be made. The provided total-c-Src antibody can be used as a positive control to demonstrate that the cells contain c-Src, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, you can prepare many plates simultaneously and then perform the assay when desired.
c-Src (proto-oncogene tyrosine-protein kinase Src) is a 60 kDa tyrosine protein kinase that contains both SH2 and SH3 domains. c-Src is a lipid-modified signaling molecule (myristoylated) involved in cell-cell interactions, cell migration and proliferation through the phosphorylation of many substrates. c-Src is expressed at high levels in differentiated cells including neurons, platelets and macrophages, and can be activated by phospholipase D, insulin-like growth factor receptor, EGF-receptor, fibroblast growth factor receptor and prolactin receptor binding. c-Src can be phosphorylated on multiple sites to interact with polyoma virus middle T antigen, PYK2, HSP72 and caveolin, and efficient study methods are in high demand.
The Src gene family is represented by at least eight different protein tyrosine kinases that belong to the non-receptor tyrosine kinase family. These protein tyrosine kinases are important regulators of many cellular processes, including cytoskeletal organization, cell-cell contact, DNA synthesis and cellular proliferation. Members of this group of proteins include c-Src, c-Yes, Fyn, Lck, Lyn, Hck, Blk and c-Fgr. The proto-oncogene c-Src is the prototype member of this gene family and is expressed in a broad range of tissues and cells. Elevated c-Src tyrosine kinase activity has been found in many types of human cancers, most notably in breast carcinomas.
The activity of Src family tyrosine kinase (SFK) is determined by an equilibrium between an inactive (phosphorylated) and a primed (dephosphorylated) state regulated by a balance of Csk (C-terminal Src kinase) and its counteracting tyrosine phosphatase(s). Upon cell stimulation, SFKs at a primed state become functionally activated to relay signals into the cells. Thus, perturbation of the equilibrium status of SFK may greatly affect the sensitivity of the cells to extracellular cues.
There are two tyrosine phosphorylation sites with opposing effects in Src. Tyrosine 418 is located in the catalytic SH2 domain and is one of the autophosphorylation sites. Phosphorylation of Tyr418 upregulates the enzyme while phosphorylation of Tyr529 in the C-terminal tail by Csk renders the enzyme less active. SFKs are required for EGF-, PDGF- and CSF-1-stimulated entry to the S-phase of the mitotic cycle in fibroblasts. Similarly, they are activated at mitosis and play a role in cellular division at the G2-M transition phase. Activated c-Src can increase tyrosine phosphorylation of paxillin and is involved in the activation of the Ras/ERK pathway.
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Storage instructionsPlease refer to protocols.
Components 1 x 96 tests 5 x 96 tests 1% SDS Solution 1 x 22ml 5 x 22ml 10% Triton X-100 1 x 10ml 5 x 10ml 10X PBS 1 x 120ml 5 x 120ml 1X Antibody Blocking Buffer 1 x 22ml 5 x 22ml 1X Antibody Dilution Buffer 1 x 30ml 5 x 30ml 96-well tissue culture plate 2 units 10 units Anti-rabbit HRP-conjugated IgG 1 x 11µl 5 x 11µl Crystal Violet Solution 1 x 22ml 5 x 22ml Developing Solution 2 x 11ml 10 x 11ml Phospho-c-Src antibody 1 x 9µl 5 x 9µl Plate sealer 2 units 10 units Stop Solution 2 x 11ml 10 x 11ml Total-c-Src antibody 1 x 9µl 5 x 9µl
FunctionNon-receptor protein tyrosine kinase that plays pivotal roles in numerous cellular processes such as proliferation, migration, and transformation. In concert with PTK2B, plays an important role in osteoclastic bone resorption. Both the formation of a SRC-PTK2B complex, and SRC kinase activity are necessary for this function. Once it is recruited to the activated integrins, by PTK2B, it phosphorylates CBL which in turn induces the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Promotes energy production in osteoclasts by activating mitochondrial cytochrome C oxidase. Phosphorylates RUNX3 and COX2 on tyrosine residues, TNK2 on 'Tyr-284' and CBL on 'Tyr-731'. Enhances DDX58/RIG-I-elicited antiviral signaling.
Sequence similaritiesBelongs to the protein kinase superfamily. Tyr protein kinase family. SRC subfamily.
Contains 1 protein kinase domain.
Contains 1 SH2 domain.
Contains 1 SH3 domain.
modificationsDephosphorylated at Tyr-530 by PTPRJ (By similarity). Phosphorylated on Tyr-530 by c-Src kinase (CSK). The phosphorylated form is termed pp60c-src. Dephosphorylated by PTPRJ at Tyr-419. Normally maintained in an inactive conformation with the SH2 domain engaged with Tyr-530, the SH3 domain engaged with the SH2-kinase linker, and Tyr-419 dephosphorylated. Dephosphorylation of Tyr-530 as a result of protein tyrosine phosphatase (PTP) action disrupts the intramolecular interaction between the SH2 domain and Tyr-530, Tyr-419 can then become autophosphorylated, resulting in SRC activation. Phosphorylation of Tyr-530 by CSK allows this interaction to reform, resulting in SRC inactivation.
S-nitrosylation is important for activation of its kinase activity.
Cellular localizationCell membrane. Mitochondrion inner membrane.
- Information by UniProt
- Avian sarcoma virus
ab207461 has not yet been referenced specifically in any publications.