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Alexa Fluor® 488 Mouse anti-PKCα (pT497)
Alexa Fluor® 488 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 Alexa Fluor® 488 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 Alexa Fluor® 488 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
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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_1645306
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® 488 under optimum conditions, and unreacted Alexa Fluor® 488 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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. 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.
  4. Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
  5. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. 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.
  8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
560139 Rev. 3
Antibody Details
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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.

560139 Rev. 3
Format Details
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Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
560139 Rev.3
Citations & References
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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
560139 Rev. 3

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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.