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PE Mouse anti-Human PKCθ
PE Mouse anti-Human PKCθ

Analysis of PKCθ in lymphocytes.  Human peripheral blood mononuclear cells (PBMC) were either stimulated with 10 μM PMA (Sigma, Cat. No. P8139) for 24 hours (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 Mouse anti-PKCθ.  Lymphocytes were selected by scatter profile.  The data demonstrates that the expression of PKCθ decreases when the lymphocytes are stimulated by PMA.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

The specificity of mAb 27/PKCθ was confirmed by western blot analysis using unconjugated antibody (Cat. No. 610089 or 610090) at 4.0 μg/ml on lysates from control (lane 1) and PMA-treated (lane 2) PBMC.  PKCθ is identified as a band of 79 kDa, which decreases in intensity in the treated cells.

Analysis of PKCθ in lymphocytes.  Human peripheral blood mononuclear cells (PBMC) were either stimulated with 10 μM PMA (Sigma, Cat. No. P8139) for 24 hours (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 Mouse anti-PKCθ.  Lymphocytes were selected by scatter profile.  The data demonstrates that the expression of PKCθ decreases when the lymphocytes are stimulated by PMA.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

The specificity of mAb 27/PKCθ was confirmed by western blot analysis using unconjugated antibody (Cat. No. 610089 or 610090) at 4.0 μg/ml on lysates from control (lane 1) and PMA-treated (lane 2) PBMC.  PKCθ is identified as a band of 79 kDa, which decreases in intensity in the treated cells.

Product Details
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BD Phosflow™
KPCT, nPKC-θ, PRKCQ, PRKCT
Human (QC Testing)
Mouse IgG2a, κ
Human PKCθ aa. 21-217 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645526
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.

Recommended Assay Procedures

Either BD Cytofix™ Fixation Buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation. Any of the three BD Phosflow™ permeabilization buffers may be used.

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.
560216 Rev. 2
Antibody Details
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27/PKCθ

The Protein Kinase C (PKC) family of homologous serine/threonine protein kinases is involved in a number of processes, such as growth, differentiation, and cytokine secretion.  At least eleven isozymes have been described.  These proteins are products of multiple genes and alternative splicing.  Conventional PKC (cPKC) subfamily members (α, β, and γ isoforms) consists of a single polypeptide chain containing four conserved regions (C) and five variable regions (V).  The N-terminal half containing C1, C2, V1, and V2 constitutes the regulatory domain and interacts with the PKC activators Ca2+, phospholipid, diacylglycerol, or phorbol ester.  However, the the C2-like domains of novel PKC (nPKC) subfamily members (δ, ε, η, and θ isoforms) are Ca2+-independent.  The atypical PKC (aPKC) subfamily members (ζ, ι, and λ isoforms) lack the C2 domain and are unique in that their activity is independent of diacylglycerols and phorbol esters. They also lack one repeat of the cysteine-rich sequences that are conserved in cPKC and nPKC members.  The C-terminal region of PKC contains the catalytic domain.  The PKC pathway represents a major signal transduction system that is activated following ligand-stimulation of transmembrane receptors by hormones, neurotransmitters, and growth factors.  PKCθ transcripts are expressed in most tissues with the highest levels being found in hematopoietic tissues and cell lines, including T cells and thymocytes.  PKCθ mRNA is readily detectable in skeletal muscle, lung and brain.  However, PKCθ expression is not detected in several human carcinoma cell lines.  Abundant expression of this PKC isozyme in hematopoietic cells suggest that it may have a role in growth and differentiation processes of these cells.

The 27/PKCθ monoclonal antibody recognizes the C2-like domain of human PKCθ, regardless of phosphorylation status.

560216 Rev. 2
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.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560216 Rev.2
Citations & References
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Development References (4)

  1. Manicassamy S, Gupta S, Sun Z. Selective function of PKC-θ in T cells. Cell Mol Immunol. 2006; 3(4):263-270. (Biology). View Reference
  2. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988; 334(6184):661-665. (Biology). View Reference
  3. Parker PJ, Murray-Rust J. PKC at a glance. J Cell Sci. 2004; 117:131-132. (Biology). View Reference
  4. Soderling TR. Protein kinases. Regulation by autoinhibitory domains. J Biol Chem. 1990; 265(4):1823-1826. (Biology). View Reference
View All (4) View Less
560216 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.