17-10060 | ChIPAb+ NFκB p65 (RelA) - ChIP Validated Antibody and Primer Set

Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from serum starved, TNFα-treated (20 ng/mL, 30 min) HeLa cells (~3 X 106 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 µg of either Normal Mouse IgG or 4 µg of Anti-NFκB p65 (RelA) and the Magna ChIP™ G Kit (Cat. # 17-611). Successful immunoprecipitation of NFκB p65 (RelA) associated DNA fragments was verified by qPCR using ChIP Primers, IĸBα promoter (Figure 1). Please refer to the EZ-Magna ChIP™ G (Cat. # 17-409) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared fro
17-10060
25  25 assays per set. Recommended use: ~4 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).
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      Overview

      Key Spec Table

      Species ReactivityKey Applications
      R, H, MChIP, WB
      Description
      Catalogue Number 17-10060
      Brand Family Upstate
      Synonyms Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 Transcription factor p65 Nuclear factor NF-kappa-B p65 subunit
      Trade Name
      • Upstate
      • ChIPAb+
      Description ChIPAb+ NFκB p65 (RelA) - ChIP Validated Antibody and Primer Set
      Overview All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction.
      The ChIPAb+ NFκB p65 (RelA) set includes the NFκB p65 (RelA) antibody, a negative control normal mouse IgG, and qPCR primers which amplify a 299 bp region of human IκBα promoter. The NFκB p65 (RelA) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of NFκB p65 (RelA)-associated chromatin.
      Background Information The transcription factor NFkappaB (Nuclear Factor kappa B) is involved in the expression and regulation of a number of important cellular and physiological processes such as growth, development, apoptosis, immune and inflammatory response, and activation of various viral promoters including human immunodeficiency virus long terminal repeats. NFkappaB represents a group of structurally related and evolutionarily conserved proteins related to the proto-oncogene c-Rel with five members in mammals that include Rel (cRel), RelA (p65), RelB, NFkappaB1 (p50 and its precursor p105), and NFkappaB2 (p52 and its precursor p100). NFkappaB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes. Although most NFkappaB dimers are activators of transcription, the p50/50 and p52/52 homodimers can repress the transcription of their target genes. The p50/p65 heterodimer of NFkappaB is the most abundant in cells.
      References
      Product Information
      Format Purified
      Specificity This antibody recognizes an epitope overlapping the nuclear location signal (NLS) of the p65 subunit of the NFkB heterodimer. Thus it selectively binds to the activated form of NFkB.
      Control
      • Includes negative control normal mouse IgG and primers specific for human IĸBα promoter.
      Presentation Anti-NFκB p65 (RelA) (mouse monoclonal). One vial containing 100 µg of protein A purified monoclonal IgG3 in a total volume of 143 uL in a buffer containing 0.02 M phosphate buffer, 0.25 M NaCl, pH 7.6 with 0.09% sodium azide, before the addition of glycerol to 30%. Store at -20°C.

      Nornal Mouse IgG. One vial containing 125 µg of purified mouse IgG in 125 µL of storage buffer containing 0.1% sodium azide. Store at -20°C.

      ChIP Primers, IĸBα promoter. One vial containing 75 μL of 5 μM of each primer specific for human IĸBα promoter. Store at -20°C.
      FOR: GAC GAC CCC AAT TCA AAT CG
      REV: TCA GGC TCG GGG AAT TTC C
      Applications
      Application This ChIPAb+ NFκB p65 (RelA) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
      Key Applications
      • Chromatin Immunoprecipitation (ChIP)
      • Western Blotting
      Application Notes Chromatin Immunoprecipitation:
      Representative lot data.
      Sonicated chromatin prepared from serum starved, TNFα-treated (20 ng/mL, 30 min) 293 cells (~3 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 µg of Normal Mouse IgG or 4 µg of Anti-NFκB p65 (RelA) and the Magna ChIP™ A Kit (Cat. # 17-610).
      Successful immunoprecipitation of NFκB p65 (RelA) associated DNA fragments was verified by qPCR using ChIP Primers, IĸBα promoter as a positive locus, and β-Actin promoter primers as a negative locus (Please see figures). Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.
      Please refer to the EZ-Magna ChIP™ A (Cat. # 17-409) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.


      Western Blot Analysis:
      Representative lot data.
      Huvec lysate (Lane 1), L6 lysate (Lane 2) and PC12 lysate (Lane 3) were resolved by electrophoresis, transferred to PVDF membrane and probed with anti-NFκB p65 (RelA) (1:500 dilution).
      Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a chemiluminescence detection
      system.
      Arrows indicates protein NFκB p65 (RelA) (~65 kDa) (Please see figures).
      A non-specific band may be seen at ~230 kDa in L6 lysate.

      Immunofluorescence: A 1-10 μg/mL concentration of a previous lot was used in immunofluorescence.

      Immunohistochemistry (paraffin sections): A 5-10 μg/mL (APAAP)
      concentration of a previous lot was used in immunohistochemistry.

      Immunohistochemistry (frozen sections): A 5-10 μg/mL (APAAP)
      concentration of a previous lot was used in immunohistochemistry.

      Electrophoretic Mobility Shift Assay (EMSA): A 0.5-1 μg/mL concentration of a previous lot was used in shift assay.

      Flow Cytometry: A previous lot of this antibody was used in flow cytometry.

      Optimal working dilutions must be determined by end user.
      Biological Information
      Immunogen species Peptide corresponding to human p65 coupled to BSA.
      Epitope NLS of p65 subunit
      Host Mouse
      Specificity This antibody recognizes an epitope overlapping the nuclear location signal (NLS) of the p65 subunit of the NFkB heterodimer. Thus it selectively binds to the activated form of NFkB.
      Isotype IgG3
      Species Reactivity Rat Human Mouse
      Species Reactivity Note Human and rat. Expected to react with mouse based on sequence homology.
      Antibody Type Monoclonal Antibody
      Entrez Gene Number
      Entrez Gene Summary The p50 (NFKB1)/p65 (RELA) heterodimer is the most abundant form of NFKB. The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA, MIM 164008 or NFKBIB, MIM 604495), which inactivate NFKB by trapping it in the cytoplasm. Phosphorylation of serine residues on the I-kappa-B proteins by kinases (IKBKA, MIM 600664, or IKBKB, MIM 603258) marks them for destruction via the ubiquitination pathway, thereby allowing activation of the NFKB complex. Activated NFKB complex translocates into the nucleus and binds DNA at kappa-B-binding motifs such as 5-prime GGGRNNYYCC 3-prime or 5-prime HGGARNYYCC 3-prime (where H is A, C, or T; R is an A or G purine; and Y is a C or T pyrimidine).
      Gene Symbol
      • p50/p65
      • RELA
      • NFKB
      • p65
      Purification Method Protein A purfied
      UniProt Number
      UniProt Summary Function: NF-kappa-B is a pleiotropic transcription factor which is present in almost all cell types and is involved in many biological processed such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. NF-kappa-B heterodimeric p65-p50 and p65-c-Rel complexes are transcriptional activators. The NF-kappa-B p65-p65 complex appears to be involved in invasin-mediated activation of IL-8 expression. The inhibitory effect of I-kappa-B upon NF-kappa-B the cytoplasm is exerted primarily through the interaction with p65. p65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex.

      Subunit structure: Component of the NF-kappa-B p65-p50 complex. Component of the NF-kappa-B p65-c-Rel complex. Homodimer; component of the NF-kappa-B p65-p65 complex. Component of the NF-kappa-B p65-p52 complex. May interact with ETHE1. Binds AES and TLE1. Interacts with TP53BP2. Binds to and is phosphorylated by the activated form of either RPS6KA4 or RPS6KA5. Interacts with ING4 and this interaction may be indirect. Interacts with CARM1, USP48 and UNC5CL. Interacts with IRAK1BP1. Interacts with NFKBID. Interacts with NFKBIA. Interacts with GSK3B. Interacts with NFKBIB. Interacts with NFKBIE. Interacts with NFKBIZ. Part of a 70-90 kDa complex at least consisting of CHUK, IKBKB, NFKBIA, RELA, IKBKAP and MAP3K14. Interacts with HDAC3; HDAC3 mediates the deacetylation of RELA. Interacts with HDAC1; the interaction requires non-phosphorylated RELA. Interacts with CBP; the interaction requires phosphorylated RELA. Interacts (phosphorylated at 'Thr-254') with PIN1; the interaction inhibits p65 binding to NFKBIA. Interacts with SOCS1. Interacts with UXT. Interacts with MTDH and PHF11. Interacts with ARRB2. Interacts with human respiratory syncytial virus (HRSV) protein M2-1. Interacts with NFKBIA (when phosphorylated), the interaction is direct; phosphorylated NFKBIA is part of a SCF(BTRC)-like complex lacking CUL1.

      Subcellular location: Nucleus. Cytoplasm. Note: Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B).

      Post-translational modification: Ubiquitinated, leading to its proteosomal degradation. Degradation is required for termination of NF-kappa-B response.
      Phosphorylation on 'Ser-536' stimulates acetylation on 'Lys-310' and interaction with CBP; the phosphorylated and acetylated forms show enhanced transcriptional activity.
      Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3. Acetylation at 'Lys-122' enhances DNA binding and impairs association with NFKBIA. Acetylation at 'Lys-310' is required for full transcriptional activity in the absence of effects on DNA binding and NFKBIA association. Acetylation can also lower DNA-binding and results in nuclear export.

      Sequence similarities: Contains 1 RHD (Rel-like) domain.
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality Assurance Chromatin Immunoprecipitation:
      Sonicated chromatin prepared from serum starved, TNFα-treated (20 ng/mL, 30 min) 293 cells (~3 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 µg of either Normal Mouse IgG or 4 µg of Anti-NFκB p65 (RelA) and the Magna ChIP™ A Kit (Cat. # 17-610).
      Successful immunoprecipitation of NFκB p65 (RelA) associated DNA fragments was verified by qPCR using ChIP Primers, IĸBα promoter (Please see figures).
      Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage Conditions Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance. Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.
      Packaging Information
      Packaging 25 assays per set. Recommended use: ~4 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).
      Qty/Pck 25 assays
      Material Size 25
      Material Package 25 assays per set. Recommended use: ~4 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      Certificates of Analysis

      TitleLot Number
      ChIPAb+ NFB p65 (RelA) - 2268797 2268797
      ChIPAb+ NFκB p65 (RelA) 2465307
      ChIPAb+ NFκB p65 (RelA) - 2376470 2376470
      Millipore - COA - ChIPAb+ NF#954;B p65 (RelA) - NG1850740 NG1850740
      Millipore - COA - ChIPAb+ NFB p65 (RelA) - 1973238 1973238
      Millipore - COA - ChIPAb+ NFB p65 (RelA) - 2053032 2053032
      Millipore - COA - ChIPAb+ NFB p65 (RelA) - 2142728 2142728
      Millipore - COA - ChIPAb+ NFB p65 (RelA) - NG1880233 NG1880233
      Millipore - COA - ChIPAb+ NFB p65 (RelA) - NG1905709 NG1905709
      Millipore - COA - ChIPAb+ NFB p65 (RelA) - NRG1768684 NRG1768684