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      PE-Cy™7 Mouse anti-NF-κB p65 (pS529)
      PE-Cy™7 Mouse anti-NF-κB p65 (pS529)
      Analysis of NFkB (pS529) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either stimulated with 50 nM PMA (Sigma, P8139) for 15 minutes (shaded histogram) or unstimulated (open histogram). The PBMC were fixed with BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes and then stained with PE-Cy™7 Mouse anti-NF-κB p65 (pS529). For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
      PE-Cy™7 Mouse anti-NF-κB p65 (pS529) PE-Cy™7 Mouse anti-NF-κB p65 (pS529)
      Analysis of NFkB (pS529) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either stimulated with 50 nM PMA (Sigma, P8139) for 15 minutes (shaded histogram) or unstimulated (open histogram). The PBMC were fixed with BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes and then stained with PE-Cy™7 Mouse anti-NF-κB p65 (pS529). For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
      Product Details
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      BD Phosflow™
      RELA; REL-A; NFKBp65; NFKB3; Transcription factor p65; TF65
      Human (QC Testing)
      Mouse BALB/c IgG2b, κ
      Phosphorylated Human NF-κB p65 Peptide
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      AB_1645545
      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 PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed.
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      Recommended Assay Procedures

      This antibody conjugate is suitable for intracellular staining of human cell lines and peripheral blood mononuclear cells using BD Cytofix™ Fixation Buffer.  Any of the three BD Phosflow™ permeabilization buffers may be used.

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      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. PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
      4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      5. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
      6. Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
      7. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      8. 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.
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      560335 Rev. 2
      Antibody Details
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      K10-895.12.50

      The K10-895.12.50 monoclonal antibody recognizes the phosphorylated serine 529 (pS529) in the transactivation domain of the human NF-κB p65 subunit. Nuclear factor κB (NF-κB) is a ubiquitously expressed transcription factor that regulates the expression of many other genes. It is crucial for cellular responses to a variety of stimuli including stress and microbial pathogens that lead to immunity, inflammation, proliferation, differentiation, survival, apoptosis, and tumorigenesis. The most studied NF-κB complex consists of the p50 (also known as NF-κB1) and p65 (also known as REL-A) subunits, both containing a 300-amino acid region with homology to the Rel proto-oncogene product (RH domain). The RH domain contains motifs for dimerization, nuclear localization, and binding to specific DNA sequences. In addition to the RH domain, the p65 subunit contains the transactivation domain, which is responsible for the interaction with the inhibitor IκB and which contains phosphorylation sites. In most cell types, the p50/p65 heterodimer is located within the cytoplasm complexed to IκB. This complex prevents nuclear translocation and activity of NF-κB. In response to stimuli such as cytokines, LPS, DNA damage, and microbial infections, IκB is phosphorylated at critical residues. This phosphorylation induces dissociation of the IκB/NF-κB complex, allowing the free heterodimeric NF-κB to translocate to the nucleus. Furthermore, optimal activation of NF-κB requires phosphorylation in the transactivation domain of p65. In the nucleus, activated NF-κB dimers bind to the κB sites within promoters and enhancers and function as transcriptional regulators.

      560335 Rev. 2
      Format Details
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      PE-Cy7
      PE-Cy7 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 781-nm. PE can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 781 nm (e.g., a 760/60-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the Red (627–640-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
      altImg
      PE-Cy7
      Yellow-Green 488 nm, 532 nm, 561 nm
      496 nm, 566 nm
      781 nm
      560335 Rev.2
      Citations & References
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      View product citations for antibody "560335" on CiteAb

      Development References (3)

      1. Natoli G, Saccani S, Bosisio D, Marazzi I. Interactions of NF-kappaB with chromatin: the art of being at the right place at the right time. Nat Immunol. 2005; 6(5):439-445. (Biology). View Reference
      2. Siebenlist U, Brown K, Claudio E. Control of lymphocyte development by nuclear factor-kappaB. Nat Rev Immunol. 2005; 5:435-445. (Biology). View Reference
      3. Viatour P, Merville M-P, Bours V, Chariot A. Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci. 2005; 30(1):43-52. (Biology). View Reference
      560335 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.