Invitrogen™ Phospho-SRC (Tyr418) Monoclonal Antibody (SC1T2M3), eBioscience™, Invitrogen™

Catalog No.	Quantity
14-903-482	100 μg
14-903-437	2 mg
14-903-495	10 mg

Catalog No. 14-903-482 Supplier Invitrogen™ Supplier No. 14903482

Description

Mouse Monoclonal Antibody

This SC1T2M3 MAb recognizes human and mouse Src tyrosine kinase (also known as ASV, c-src, c-SRC, p60-Src, pp60c-src, Proto-oncogene c-Src, Proto-oncogene tyrosine-protein kinase Src, SRC1) when phosphorylated on tyrosine 418 (Y418). Autophosphorylation of Src at Y418 in the catalytic domain is required for full catalytic activity of this kinase. Src is a non-receptor tyrosine kinase involved in signal transduction in numerous biological systems and is activated following engagement of many different classes of cellular receptors including immune response receptors, integrins and other adhesion receptors, receptor protein tyrosine kinases, G protein-coupled receptors as well as cytokine receptors. Aberrant Src activity has been implicated in the development of numerous types of cancer. Due to the sequence homology surrounding Src Y418, this SC1T2M3 clone is predicted to cross-react with many Src family kinases including Src, Lck, Fyn, and Lyn. Specificity of this SC1T2M3 clone was determined by ELISA, flow cytometry, and western blotting.

SRC is a cytoplasmic tyrosine kinase and proto-oncogene involved in the regulation of embryonic development and cell growth. SRC is involved in the regulation of growth and differentiation of eukaryotic cells. SRC associates with the cytoplasmic region of various cell surface receptors to facilitate signal transduction, and is also involved in cytoskeletal organization, cell cycle regulation, and apoptosis. In humans, the gene encoding SRC is present on chromosome 20. Activity of SRC is regulated by two tyrosine residues (Tyr 416 and Tyr527), and phosphorylation of these two sites have opposing effects. Phosphorylation of Tyr416 in the activation loop of the kinase domain upregulates enzyme activity while the phosphorylation of Tyr527 decreases enzyme activity. Mutations in the SRC gene could be involved in the malignant progression of colon cancer. Two transcript variants encoding the same SRC protein have been found thus far.

Specifications

• Antigen: Phospho-SRC (Tyr418)
• Applications: Western Blot
• Classification: Monoclonal
• Clone: SC1T2M3
• Concentration: 0.5 mg/mL
• Conjugate: Unconjugated
• Formulation: PBS with 0.09% sodium azide; pH 7.2
• Gene: SRC
• Gene Accession No.: P05480, P12931
• Gene Alias: ASV; AW259666; csrc; c-SRC; c-src protein, C terminus; EC 2.7.10.2; fc54g04; Neuronal proto-oncogene tyrosine-protein kinase Src; non-tyrosine protein kinase; ORF; OTTHUMP00000174476; OTTHUMP00000174477; OTTHUMP00000174478; p60-Src; p60-Src-1; PP60C-SCR; pp60c-src; pp60src; pp60v-src; protein-tyrosine kinase; proto-oncogene c-Src; protooncogene SRC, Rous sarcoma; Proto-oncogene tyrosine-protein kinase Src; Rous sarcoma oncogene; RP5-823N20.1; sb:cb864; SDR; src; src downstream region; src oncogene; SRC proto-oncogene, non-receptor tyrosine kinase; SRC proto-oncogene, non-receptor tyrosine kinase L homeolog; SRC proto-oncogene, non-receptor tyrosine kinase S homeolog; src.L; src.S; SRC1; src-1; src1-A; src-a; src-b; THC6; transforming protein; tyrosine kinase pp60c-src; tyrosine protein kinase c-src; tyrosine protein kinase pp60-c-src; tyrosine-protein kinase SRC-1; Unknown; v-src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog; v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog; v-src sarcoma viral oncogene; wu:fc54g04; XELAEV_18046582mg; xsrc
• Gene Symbols: SRC
• Host Species: Mouse
• Purification Method: Affinity chromatography
• Quantity: 100 μg
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 20779, 6714
• Target Species: Human, Mouse
• Content And Storage: 4°C
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG2b κ

Frequently Asked Questions (FAQs)

How does eBioscience determine the specificity of their phosphorylation-specific antibodies?

Antibodies are tested and validated in a variety of ways. First, we verify that in a panel of cells in which different pathways have been induced, we see phosphorylation specific staining only in the cells in which the specific pathway of interest has been activated. Second, we verify that phosphorylation specific staining is observed only in cell types in which the protein is expressed and not in cell types in which the protein is not expressed. Third, whenever possible, western immunoblotting is used to confirm the presence of a band(s) of the appropriate size(s) in stimulated/treated cells (and not in unstimulated/untreated cells, as appropriate).

How long can cells that have been fixed and placed in methanol be stored for use with eBioscience antibodies? What is the recommended storage temperature? How about storage of fixed cells in eBioscience fixation buffers?

We have data demonstrating that fixed cells stored in methanol are stable at -20 degrees C or at -80 degrees C for several weeks. We do not recommend storing fixed cells in eBioscience Foxp3/Transcription Factor Buffer. However, cells fixed with eBioscience Foxp3/Transcription Factor Buffer or eBioscience Intracellular (IC) fixation buffer can be stored in eBioscience IC fixation buffer for up to 3 days at 4 degrees C in the dark.
Note: Please be aware that higher compensation values may be seen with tandem dyes if any other fixative is used apart from eBioscience IC fixation buffer, for 3-day storage.

What is the optimal protocol for eBioscience phosphorylation-specific antibodies?

Each antibody has been tested in three different buffer systems: IC fixation and permeabilization, Foxp3/Transcription Factor Buffer, and IC fixation Buffer/Methanol. The recommended buffer system(s) will be noted on the Technical Data Sheet for the specific antibody.

What is the best way to maintain surface staining when using the methanol-based protocol?

Because methanol can destroy the epitope recognized by some antibodies, ideally surface staining would be performed at the beginning of the protocol with a methanol-resistant fluorochrome (like eFluor 450, FITC, and eFluor 660).
Please recognize that surface staining before stimulation can have undesired effects due to activation of the cell caused by antibody binding. We recommend staining after stimulation/treatment, fixation, and methanol treatment.

Can I use other buffer systems from other companies when using eBioscience phosphorylation-specific antibodies?

In general, you can use other companies' buffer systems provided that their buffers are of a similar composition as the buffers recommended. Be aware that results will vary depending upon the buffer system used. eBioscience antibodies have been optimized for use in eBioscience buffers and we have not tested all of our antibodies in other buffer systems. Thus, we highly recommend using our optimized protocol and buffer systems.

What are the appropriate controls to run when using eBioscience phosphorylation-specific antibodies?

It is important to understand whether the stimulation/treatment results in an upregulation or a down regulation of the phosphorylation event. Thus, it is recommended that changes in phosphorylation levels in stimulated/treated versus unstimulated/untreated cells be compared. A histogram or density plot overlay these two treatments will provide a better assessment of a change in phosphorylation event than using isotype controls.

Can I perform phosphorylation specific staining and stain for intracellular cytokines at the same time when using eBioscience phosphorylation-specific antibodies?

In theory, if the buffer systems are compatible for the antibodies of interest, this can be done. In practice, cytokine translation and phosphorylation events do not typically occur in the same timeframe, therefore, it may be difficult to detect both events in the same sample. The results will be depended on the kinetics and should be determined for each system and protein of interest.

When using eBioscience phosphorylation-specific antibodies, can I perform phosphorylation specific staining together with transcription factor staining at the same time? If so, what buffer and protocol do I use?

In theory, this can be done if the antibodies against both the transcription factor and the phosphorylated protein work in the same buffer system. For example, Anti-Human/Mouse phsopho-H2AX and all transcription factor eBioscience antibodies offered will work in the Foxp3/Transcription Factor Buffer System (Cat. No. 00-5523-00).

For eBioscience phosphorylation-specific antibodies, can total and phosphorylated protein be analyzed in parallel?

In theory, this can be done if the antibodies to both proteins work in the same buffer system and if the antibodies recognize different epitopes. Please refer to the individual Technical Data Sheets for information about buffer compatibility. Also, consider using eBioscience InstantOne ELISA Kits for immunoassay quantitation of both total and phosphorylated proteins such as AKT1/2/3, JNK1/2/3, NFκB, STAT3, and STAT5.