Human Phospho-Src (Y419) DuoSet IC ELISA
Human Phospho-Src (Y419) DuoSet IC ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15- (96-well) plate pack sizes
- Economical alternative to Western blot
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
The Human Phospho-Src (Y419) DuoSet® IC ELISA specifically recognizes human Src phosphorylated at Y419 as determined by peptide competition. A lysate prepared from MDA-MB-468 human breast cancer cells treated with 50 mM sodium pervanadate for 20 minutes was analyzed with this ELISA. The Human Phospho-Src (Y419) Detection Antibody was either untreated (no peptide) or preincubated with a phospho-peptide containing the phospho-Src Y419 phosphorylation site, a phospho-peptide containing the Ret Y905 phosphorylation site, a phospho-peptide containing the Tie-2 Y1100 phosphorylation site, or a phospho-peptide containing the PP2A Y307 phosphorylation site. Peptides were used at 40 ng/mL. Only the phospho-peptide containing the Src Y419 phosphorylation site blocked the signal, indicating that the ELISA is specific for human Src phosphorylation.
Amounts of human phosphorylated Src, as quantified by the Human Phospho-Src (Y419) DuoSet® IC ELISA, are consistent with the relative amounts of phosphorylated Src determined by qualitative Western Blot analysis. Quantification of phosphorylated Scr (Y419) in pervabadate-treated human MDA-MB-468 cells. Lysates prepared from MDA-MB-468 human breast cancer cells treated with 50 mM sodium pervanadate for the indicated times were quantified with this DuoSet® IC ELISA. The same lysates were also immunoblotted (inset) with either Human Phospho-Src (Y419) Polyclonal Antibody (Catalog # AF2685) or Human/Mouse/Rat Src Polyclonal Antibody (Catalog # AF3389). The DuoSet® IC ELISA results are consistent with the relative amounts of phosphorylated Src family members detected by Western Blot. The immunoblot with anti-Src antibody indicated that total levels of human Src remained constant during incubation with sodium pervanadate.
Preparation and Storage
Src and other Src family protein tyrosine kinases are proto-oncogenes that play key roles in regulating cell growth and differentiation. Src family activity is regulated by tyrosine phosphorylation at two sites with opposing effects. Phosphorylation at Y419 of human Src up-regulates kinase activity, while phosphorylation at Y530 inactivates Src.
Citations for Human Phospho-Src (Y419) DuoSet IC ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 2
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Pharmacophore-based virtual screening for the identification of the novel Src inhibitor SJG-136 against lung cancer cell growth and motility
Authors: CW Weng, JH Li, JY Tsai, SH Lin, GC Chang, CC Liu, JJ Chen
Am J Cancer Res, 2020-06-01;10(6):1668-1690.
Sample Types: Cell Culture Lysates
Microarray-based kinetic colorimetric detection for quantitative multiplex protein phosphorylation analysis.
Authors: Holenya P, Kitanovic I, Heigwer F, Wolfl S
Sample Types: Recombinant Protein
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