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Alexa Fluor® 647 Mouse anti-PLC-γ2
Alexa Fluor® 647 Mouse anti-PLC-γ2

Analysis of PLC-γ2 in human peripheral blood lymphocytes.  Human whole blood was lysed and fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10-15 minutes at 37ºC, then permeabilized (BD Phosflow™ Perm Buffer II, Cat. No. 558052) on ice for 30 minutes, and then stained with Alexa Fluor® 488 Mouse Anti-Human CD3 (Cat. No. 557694), PerCP-Cy™5.5 anti-human CD20 (Cat. No. 558021), and either Alexa Fluor® 647 Mouse anti-PLC-γ2 (Cat. No. 560136; solid-line histograms) or Alexa Fluor® 647 Mouse IgG1, κ Isotype control (Cat. No. 557783, dashed line histograms).  The figures show lymphocyte subpopulations that were selected by their scatter profile and surface antigen expression.  PLC-γ2 expression on CD20-positive CD3-negative B lymphocytes (left panel) and CD20-negative CD3-negative NK cells (right panel) are displayed.  There was little detectable expression of PLC-γ2 in the CD20-negative CD3-positive T lymphocytes (not shown).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.  

Analysis of PLC-γ2 in human peripheral blood lymphocytes.  Human whole blood was lysed and fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10-15 minutes at 37ºC, then permeabilized (BD Phosflow™ Perm Buffer II, Cat. No. 558052) on ice for 30 minutes, and then stained with Alexa Fluor® 488 Mouse Anti-Human CD3 (Cat. No. 557694), PerCP-Cy™5.5 anti-human CD20 (Cat. No. 558021), and either Alexa Fluor® 647 Mouse anti-PLC-γ2 (Cat. No. 560136; solid-line histograms) or Alexa Fluor® 647 Mouse IgG1, κ Isotype control (Cat. No. 557783, dashed line histograms).  The figures show lymphocyte subpopulations that were selected by their scatter profile and surface antigen expression.  PLC-γ2 expression on CD20-positive CD3-negative B lymphocytes (left panel) and CD20-negative CD3-negative NK cells (right panel) are displayed.  There was little detectable expression of PLC-γ2 in the CD20-negative CD3-positive T lymphocytes (not shown).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.  

Product Details
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BD Phosflow™
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG1, κ
Phosphorylated Human PLC-γ2 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645321
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.

Recommended Assay Procedures

This PLC-γ2-specific antibody conjugate may be used with conjugates of anti-PLC-γ2 (pY759) mAb K86-689.37 to distinguish the expression of total versus phosphorylated PLC-γ2.

This antibody conjugate is suitable for intracellular staining of human whole blood and mouse splenocytes using the BD Phosflow™ Lyse/Fix Buffer and the BD Phosflow™ Perm Buffer II.

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. An isotype control should be used at the same concentration as the antibody of interest.
  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. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  6. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  7. 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.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Cy is a trademark of GE Healthcare.
  10. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  11. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560136 Rev. 3
Antibody Details
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K86-1161

The Phospholipase C (PLC) isozymes hydrolyze phosphatidyl inositol biphosphate to inositol triphosphate and diacylglycerol.  The former causes release of calcium from the endoplasmic reticulum, while the latter is an activator of Protein Kinase C.  Within the PLC family, PLC-γ is the only member that contains SH2 and SH3 domains.  These domains enable it to interact with receptor tyrosine kinases and become enzymatically activated via phosphorylation.  It exists as two isoforms: 1) PLC-γ1, which is ubiquitously expressed, and 2) PLCγ2, found primarily in the lymphoid system.  PLC-γ is essential for growth factor-induced cell motility and mitogenesis.  Overexpression of PLC-γ is evident in several forms of cancer, and it has been identified as a key mediator of PDGF-dependent cellular transformation.  Thus regulation of PLC-γ activity by growth factors is involved in cell growth and transformation.

Although the immunogen for generation of the K86-1161 monoclonal antibody was a phosphorylated peptide, peptide blocking studies demonstrated that the mAb recognizes PLC-γ2 regardless of phosphorylation status.  This antibody was raised to a unique region of PLC-γ2 and is predicted not to crossreact with PLC-γ1

560136 Rev. 3
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
560136 Rev.3
Citations & References
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Development References (5)

  1. Kim YJ, Sekiya F, Poulin B, Bae YS, Rhee SG. Mechanism of B-cell receptor-induced phosphorylation and activation of phospholipase C-γ2. Mol Cell Biol. 2004; 24(22):9986-9999. (Biology).
  2. Knoll M, Yanagisawa Y, Simmons S, et al. The non-Ig parts of the vpreB and λ5 proteins of the surrogate light chain play opposite roles in the surface representation of the precursor b cell receptor. J Immunol. 2012; 188(12):6010-6017. (Clone-specific: Flow cytometry). View Reference
  3. Ozdener F, Dangelmaier C, Ashby B, Kunapuli SP, Daniel JL. Activation of phospholipase Cγ2 by tyrosine phosphorylation.. Mol Pharmacol. 2002; 62(3):672-679. (Biology).
  4. Wang D, Feng J, Wen R, et al. Phospholipase Cγ2 is essential in the functions of B cell and several Fc receptors. Immunity. 2000; 13:25-35. (Biology).
  5. Wen R, Jou S-T, Chen Y, Hoffmeyer A, Wang D. Phospholipase Cγ2 is essential for specific functions of FcεR and FcγR . J Immunol. 2002; 169:6743-6752. (Biology).
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560136 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.