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Alexa Fluor® 647 Mouse anti-p130Cas (pY249)
Alexa Fluor® 647 Mouse anti-p130Cas (pY249)

Analysis of p130Cas (pY249) in activated human Burkitt's lymphoma.  Ramos cells were serum starved overnight and then either stimulated with 5 mM hydrogen peroxide for 15 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Phosflow™ Fix Buffer I, Cat. No. 557870) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-p130Cas (pY249, Cat. No. 558547).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Analysis of p130Cas (pY249) in activated human Burkitt's lymphoma.  Ramos cells were serum starved overnight and then either stimulated with 5 mM hydrogen peroxide for 15 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Phosflow™ Fix Buffer I, Cat. No. 557870) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-p130Cas (pY249, Cat. No. 558547).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Phosflow™
Human (QC Testing)
Mouse IgG2b, κ
Phosphorylated Human p130Cas
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_647137
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  5. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558547 Rev. 2
Antibody Details
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J169-757.12.2

p47v-crk (v-Crk) is the product of a transforming gene, v-crk, that was isolated from avian sarcoma viruses.  The v-Crk protein is a fusion product of viral Gag protein and a part of cellular Crk that includes SH2 and SH3 domains.  v-Crk-induced transformation increases tyrosine phosphorylation of several cellular proteins, including p130Cas (CRK-associated substrate).  The p130Cas is tightly associated with v-Crk via the SH2 domain of v-Crk.  Tyrosine phosphorylation of p130Cas occurs in conjunction with cellular transformation in cells that express v-Src or v-Crk.  This phosphorylation leads to a change in p130Cas localization from the cytoplasm to the cell membrane and, possibly, to the nucleus.  Since p130Cas also associates with v-Src, it may be a v-Src substrate.  Several phosphorylation sites have been described in p130Cas upon Fibroblast Growth Factor stimulation, and phosphorylated tyrosine (Y249) might function as a binding site for the Crk-adaptor molecule.

The J169-757.12.2 monoclonal antibody recognizes the phosphorylated Y249 of human p130Cas.  The orthologous phosphorylation sites in mouse and rat p130Cas are Y253 and Y347, respectively.

558547 Rev. 2
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
558547 Rev.2
Citations & References
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Development References (2)

  1. Goldberg GS, Alexander DB, Pellicena P, Zhang Z-Y, Tsuda H, Miller WT. Src phosphorylates Cas on tyrosine 253 to promote migration of transformed cells. J Biol Chem. 2003; 278(47):46533-46540. (Biology).
  2. Hinsby AM, Olsen JV, Bennett KL, Mann M. Signaling initiated by overexpression of the fibroblast growth factor receptor-1 investigated by mass spectrometry. Mol Cell Proteomics. 2003; 2(1):29-36. (Biology).
558547 Rev. 2

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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.