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    R718 Mouse Anti-Human NF-κB p65 (pS529)
    R718 Mouse Anti-Human NF-κB p65 (pS529)
    Flow cytometric analysis of NF-κB p65 (pS529) expression by TNF-treated HeLa S3 cells. Cultured cells from the human HeLa S3 (Cervical adenocarcinoma, ATCC CCL 2.2) cell line were starved overnight in Dulbecco's Minimal Eagle's Medium. The cells were harvested and washed with Dulbecco's Phosphate Buffered Saline. They were then either left untreated (dashed line histogram) or treated (37°C, 10 min) with Recombinant Human TNF protein (20 ng/mL; Cat. No. 554618; solid line histogram) and Calyculin A (50 nM). The cells were fixed (10 min; 37°C) with pre-warmed BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized (30 min on ice) with BD Phosflow™ Perm Buffer III (Cat. No. 558050) and washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656). The cells were then stained with BD Phosflow™ R718 Mouse Anti-Human NF-κB p65 (pS529) antibody (Cat. No. 567093).  The fluorescence histograms showing NF-κB p65 (pS529) expression were derived from gated events with the forward and side light-scatter characteristics of intact HeLa S3 cells. Flow cytometry and data analysis were performed using a BD™ LSR II Flow Cytometer System and FlowJo™ software.
    R718 Mouse Anti-Human NF-κB p65 (pS529) R718 Mouse Anti-Human NF-κB p65 (pS529)
    Flow cytometric analysis of NF-κB p65 (pS529) expression by TNF-treated HeLa S3 cells. Cultured cells from the human HeLa S3 (Cervical adenocarcinoma, ATCC CCL 2.2) cell line were starved overnight in Dulbecco's Minimal Eagle's Medium. The cells were harvested and washed with Dulbecco's Phosphate Buffered Saline. They were then either left untreated (dashed line histogram) or treated (37°C, 10 min) with Recombinant Human TNF protein (20 ng/mL; Cat. No. 554618; solid line histogram) and Calyculin A (50 nM). The cells were fixed (10 min; 37°C) with pre-warmed BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized (30 min on ice) with BD Phosflow™ Perm Buffer III (Cat. No. 558050) and washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656). The cells were then stained with BD Phosflow™ R718 Mouse Anti-Human NF-κB p65 (pS529) antibody (Cat. No. 567093).  The fluorescence histograms showing NF-κB p65 (pS529) expression were derived from gated events with the forward and side light-scatter characteristics of intact HeLa S3 cells. Flow cytometry and data analysis were performed using a BD™ LSR II Flow Cytometer System and FlowJo™ software.
    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)
    5 µl
    Aqueous buffered solution containing ≤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 the dye under optimum conditions and unreacted dye was removed.
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    Recommended Assay Procedures

    BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBead to ensure that BD CompBeads are appropriate for your specific cellular application.

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    Product Notices

    1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
    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. 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).
    5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
    6. This product is provided under an Agreement between BIOTIUM and BD Biosciences. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
    7. Alexa Fluor™ is a trademark of Life Technologies Corporation.
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    567093 Rev. 1
    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.

    The antibody was conjugated to BD Horizon™ Red 718, which has been developed exclusively by for BD Biosciences as a better alternative to Alexa Fluor™ 700. BD Horizon™ Red 718 can be excited by the red laser (628–640 nm) and, with an Em Max around 718 nm, it can be detected using a 730/45 nm filter. Due to similar excitation and emission properties, we do not recommend using R718 in combination with APC-R700 or Alexa Fluor™ 700.

    567093 Rev. 1
    Format Details
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    R718
    The BD Horizon™ Red 718 (R718) Dye is part of the BD red family of dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 695-nm and an emission maximum (Em Max) at 718-nm. Driven by BD innovation, R718 is designed to be excited by the red laser (627–640-nm) and detected using an optical filter centered near 720-nm (e.g., a 720/40-nm bandpass filter). R718 is a brighter alternative to Alexa Fluor™ 700. R718 is also a bright small molecule alternative to APC-R700 with lower spread into the APC detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
    altImg
    R718
    Red 627-640 nm
    695 nm
    718 nm
    567093 Rev.1
    Citations & References
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    Development References (8)

    1. Dominguez-Villar M, Gautron AS, de Marcken M, Keller MJ, Hafler DA. TLR7 induces anergy in human CD4+ T cells. Nat Immunol. 2015; 16(1):118-128. (Clone-specific: Flow cytometry). View Reference
    2. Feasibility study: phospho-specific flow cytometry enabling rapid functional analysis of bone marrow samples from patients with multiple myeloma. Cytometry B Clin Cytom. 2014; 86B:139-144. (Clone-specific: Flow cytometry). View Reference
    3. Mingueneau M, Kreslavsky T, Gray D, . The transcriptional landscape of alphabeta T cell differentiation. Nat Immunol. 2013; 14(6):619-632. (Clone-specific: Flow cytometry). View Reference
    4. 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
    5. Siebenlist U, Brown K, Claudio E. Control of lymphocyte development by nuclear factor-kappaB. Nat Rev Immunol. 2005; 5:435-445. (Biology). View Reference
    6. Suni MA, Maino VC. Flow cytometric analysis of cell signaling proteins. Methods Mol Biol. 2011; 717:155-169. (Clone-specific: Flow cytometry). View Reference
    7. 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
    8. van de Laar L, van den Bosch A, Boonstra A, et al. PI3K-PKB hyperactivation augments human plasmacytoid dendritic cell development and function. Blood. 2012; 120(25):4982-4991. (Clone-specific: Flow cytometry). View Reference
    View All (8) View Less
    567093 Rev. 1

    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.

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