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PE Mouse anti-c-Cbl (pY774)
PE Mouse anti-c-Cbl (pY774)

Analysis of c-Cbl (pY774) in activated human T leukemia cells.  Jurkat cells (ATCC TIB152) 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 Cytofix™ Fixation buffer, Cat. No. 554655) for 10 minutes, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE anti-c-Cbl (pY774).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Analysis of c-Cbl (pY774) in activated human T leukemia cells.  Jurkat cells (ATCC TIB152) 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 Cytofix™ Fixation buffer, Cat. No. 554655) for 10 minutes, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE anti-c-Cbl (pY774).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Phosflow™
Human (QC Testing)
Mouse IgG1, κ
Phosphorylated Human c-Cbl Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
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 with R-PE under optimum conditions, and unconjugated antibody and free PE were 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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
558102 Rev. 3
Antibody Details
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29/c-Cbl

Cbl (Casitas B-lineage lymphoma) was identified in the genome of a transforming retrovirus from a mouse pre-B lymphoma.  The cellular gene product c-Cbl is one of numerous Cbl-related proteins found in vertebrate and invertebrate organisms.  It is an 120-kDa adapter protein that contains multiple functional domains, including a RING finger motif, a tyrosine kinase-binding (TKB) domain, and a proline-rich region.  The TKB domain directly interacts with specific auto-phosphorylation sites in activated protein-tyrosine kinases (PTK).  Through the RING finger motif, c-Cbl recruits and activates an E2 ubiquitin-conjugating enzyme, thus targeting the activated PTK for protein degradation.  The proline-rich region contains SH3 domain-binding and 14-3-3 protein-binding motifs.  c-Cbl is also phosphorylated at tyrosines 700, 731, and 774 (Y774) by Syk- and Src-family kinases after the stimulation of some integrins and a wide variety of receptors for antigens, immunoglobulins, growth factors, cytokines, and hormones.  In turn, the phosphorylated Y774 site interacts with the SH2 domain of the CRK adapter protein.  The c-Cbl adapter protein is expressed in the cytoplasm in all tissues, with especially high levels of expression in hematopoietic cells.  Through its many functional sites, c-Cbl plays key roles in the positive and negative regulation of vital cell functions, including T Cell Receptor-mediated cellular immune responses.

The 29/c-Cbl monoclonal antibody recognizes the Y774-phosphorylated form of human c-Cbl.

558102 Rev. 3
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
558102 Rev.3
Citations & References
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Development References (2)

  1. Thien CBF, Langdon WY. CBL: Many adaptations to regulate protein tyrosine kinase. Nat Rev Mol Cell Biol. 2001; 2:294-307. (Biology).
  2. Tsygankov AY, Teckchandani AM, Feshchenko EA, Swaminathan G. Beyond the RING: CBL proteins as multivalent adapters. Oncogene. 2001; 20:6382-6402. (Biology).
558102 Rev. 3

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


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.