Skip to main content Skip to navigation
PE Mouse anti-PKCα (pT497)
PE Mouse anti-PKCα (pT497)

Analysis of PKCα (pT497) in human peripheral blood lymphocytes and vascular endothelium.  

LEFT PANEL: Human peripheral blood mononuclear cells were either treated with 50 nM calyculin A for 30 minutes at 37ºC (shaded histogram) or untreated (open histogram). For data analysis, lymphocytes were selected by their scatter profile.  

RIGHT PANEL: After serum starvation overnight, EA.hy926 cells (see reference Edgell, McDonald, Graham, 1983; ATCC CRL-2922) were detached with trypsin, washed, resuspended in serum-free DMEM, and either treated with 50 nM calyculin A (shaded histogram) for 30 minutes at 37°C or untreated (open histogram).  

The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with PE Mouse anti-PKCα (pT497).  The data demonstrates that the level of phosphorylation of PKCα increases when phosphatase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. BD Phosflow™ Fix Buffer I (Cat. No 557870) may be used for cell fixation.

Analysis of PKCα (pT497) in human peripheral blood lymphocytes and vascular endothelium.  

LEFT PANEL: Human peripheral blood mononuclear cells were either treated with 50 nM calyculin A for 30 minutes at 37ºC (shaded histogram) or untreated (open histogram). For data analysis, lymphocytes were selected by their scatter profile.  

RIGHT PANEL: After serum starvation overnight, EA.hy926 cells (see reference Edgell, McDonald, Graham, 1983; ATCC CRL-2922) were detached with trypsin, washed, resuspended in serum-free DMEM, and either treated with 50 nM calyculin A (shaded histogram) for 30 minutes at 37°C or untreated (open histogram).  

The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with PE Mouse anti-PKCα (pT497).  The data demonstrates that the level of phosphorylation of PKCα increases when phosphatase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. BD Phosflow™ Fix Buffer I (Cat. No 557870) may be used for cell fixation.

Product Details
Down Arrow Up Arrow


BD Phosflow™
Human (QC Testing), Mouse (Tested in Development)
Mouse BALB/c IgG1, κ
Phosphorylated Human PKCα Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645332
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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. 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.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
560141 Rev. 3
Antibody Details
Down Arrow Up Arrow
K14-984

The Protein Kinase C (PKC) family of serine/threonine protein kinases is involved in a number of processes such as growth, differentiation, and cytokine secretion.  Three categories exist, conventional PKC (cPKC), novel PKC (nPKC), and atypical PKC (aPKC).  All have C-terminal kinase domains, which are closely related to those of protein kinases A and B (Akt), and variable N-terminal regulatory domains that give them different modes of activation.  For example, cPKC (α, βI, βII, and γ isoforms) are calcium-activated, phospholipid-dependent serine/threonine-specific enzymes that can also be activated by phorbol esters.  However, nPKC (δ, ε, η, and θ isoforms) and aPKC (ζ, ι, and λ isoforms) are Ca2+-independent.  aPKC are unique in that their activity is independent of diacylglycerols and phorbol esters.  Phosphorylation at three conserved sites in the kinase domain is required for catalytic activity.  Specifically, the threonine 497 (T497) of PKCα is in the activation loop of the kinase domain and is phosphorylated by the constitutively active phosphoinositide-dependent kinase-1 (PDK-1).

The K14-984 monoclonal antibody recognizes the phosphorylated T497 in the kinase domain of human PKCα.

560141 Rev. 3
Format Details
Down Arrow Up Arrow
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.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560141 Rev.3
Citations & References
Down Arrow Up Arrow

Development References (2)

  1. Edgell C-JS, McDonald CC, Graham JB. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci U S A. 1983; 80:3734-3737. (Methodology).
  2. Newton AC. Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J. 2003; 370:361-371. (Biology). View Reference
560141 Rev. 3

Please refer to Support Documents for Quality Certificates


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.