70-510 NFκB p50/p65 EZ-TFA Transcription Factor Assay (Colorimetric)

70-510
1 plate  96 wells
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      Overview

      Replacement Information

      Key Specifications Table

      Species Reactivity
      H, M, R
      Description
      Catalogue Number70-510
      ReplacesSGT510
      Brand Family Upstate
      Trade Name
      • EZ-TFA
      • Upstate
      DescriptionNFκB p50/p65 EZ-TFA Transcription Factor Assay (Colorimetric)
      OverviewTest Principle

      The Non-Radioactive NFkB p50/p65 Transcription Factor Assay kit is provided in a 96-well format. During the assay, the Capture Probe, a double stranded biotinylated oligonucleotide containing the consensus sequence for NFkB binding (5'-GGGACTTTCC-3'), is mixed with cellular (nuclear) extract in the Transcription Factor Assay Buffer provided. When incubated together, the active form of NFkB contained in the nuclear extract binds to its consensus sequence. After incubation, the extract/probe/buffer mixture is then directly transferred to the streptavidin coated plate. The biotinylated double stranded oligonucleotide bound by active NFkB protein is immobilized and any inactive, unbound material is washed away. The bound NFkB transcription factor subunits, p50 and p65, are detected with specific primary antibodies, a Rabbit anti-NFkB p50 (active form) and a Rabbit anti-NFkB p65. An HRP-conjugated secondary antibody is then used for detection and provides sensitive colorimetric detection that can be read in a spectrophotometric plate reader. Included in the kit are positive cell extract, a non-specific double stranded oligonucleotide, and a specific competitor double stranded oligonucleotide.



      The NF kappa B Transcription Factor Assays were QC tested using nuclear extracts from human (HeLa) cells. However, because of the conservation in the NFkB DNA binding site and the fact that the antibodies contained in the kit cross react with rat and mouse NFkB, the kit is expected to work with samples from rat and mouse as well as human.



      For Research Use Only; Not for use in diagnostic procedures

      Use of this assay in NFkB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).
      Materials Required but Not Delivered• Nuclear Extraction kit or components
      • Pipettor (both single and multi-channel)
      • Pipette tips
      • Distilled water
      • Microplate reader (wavelength 450nm/650nm)
      • Rotator or Plate shaker (optional)
      • Plate cover (optional)
      Background InformationThe transcription factor NFkB (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. NFkB 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, NFkB1 (p50 and its precursor p105), and NFkB2 (p52 and its precursor p100). NFkB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes. Although most NFkB dimers are activators of transcription, the p50/50 and p52/52 homodimers can repress the transcription of their target genes2, 3. The p50/p65 heterodimer of NFkB is the most abundant in cells3. A critical component in NFkB regulation is the IkB Kinase (IKK) complex3. In a majority of unstimulated cells, the NFkB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IkB proteins. Upon stimulation by multiple inducers including viruses or cytokines, such as TNFa, IL-1, or PMA, IkBk is rapidly phosphorylated and degraded, resulting in the release of the NFkB complex, most commonly the p105/p65 heterodimer. The p105 subunit is cleaved into its active p50 form. This cleavage exposes the NLS sequence on the p50 subunit. The p50/p65 heteroduplex then translocates to the nucleus where it activates gene transcription. NFkB induces the transcription of its own inhibitor, IkBk, causing an autoregulatory mechanism of NFkB activity and generating the inactive form of NFkB. The newly formed nuclear NFkB-IkBk complexes are then exported out to the cytoplasm, thereby reestablishing the cytoplasmic pool of inactive NFkB complexes primed for another round of activation to take place. The wide variety of genes regulated by NFkB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins, and inducible effector enzymes.
      References
      Product Information
      Components
      • NFκB p65 Primary Antibody (Part No. 90274)
      • NFκB p50 Primary Antibody (Part No. 90275)
      • NFkB Capture Probe (Part No. 90269)
      • NFkB Specific Competitor Oligonucleotide (Part No. 90270)
      • TFA Negative Control Probe (Part No. 90271)
      • HeLa Whole Cell Extract, TNFα Treated (Part No. 90350)
      • EZ-TFA (Universal Transcription Factor Assay, Colorimetric) Catalog #70-500
      Applications
      ApplicationThe Non-Radioactive NFkB p50/p65 Transcription Factor Assay kit includes a positive cell extract, a non-specific double stranded oligonucleotide & a specific competitor double stranded oligonucleotide.
      Biological Information
      Species Reactivity
      • Human
      • Mouse
      • Rat
      Entrez Gene Number
      Entrez Gene SummaryNFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA, or RELB (MIM 604758) to form the NFKB complex. 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).[supplied by OMIM]
      Gene Symbol
      • NFKB1
      • NF-kappa-B
      • NFKB-p105
      • EBP-1
      • KBF1
      • DKFZp686C01211
      • MGC54151
      • NFKB-p50
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: Q04206 # p65 is a subunit of the nuclear factor kappa-B, a second messenger, which activates the transcription of a number of genes in multiple tissues. The inhibitory effect of I-kappa-B upon NF- kappa-B in 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.
      SIZE: 551 amino acids; 60219 Da
      SUBUNIT: Active NF-kappa-B is a heterodimer of an about 50 kDa DNA-binding subunit and the weak DNA-binding subunit p65. Two heterodimers might form a labile tetramer. 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.
      SUBCELLULAR LOCATION: Nucleus. Cytoplasm. Note=Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B).
      PTM: Ubiquitinated, leading to its proteosomal degradation. Degradation is required for termination of NF-kappa-B response.
      SIMILARITY: SwissProt: Q04206 ## Contains 1 RHD (Rel-like) domain.
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality AssuranceThe reagents in this kit have been optimized to reduce background signal while maintaining a high signal to noise ratio. Quality control testing is performed using the enclosed kit components while following the protocol described herein.
      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 ConditionsThe Transcription Factor Assay is a triple temperature storage assay kit.

      · Store kit components at the temperatures indicated on the labels and insert until their expiration date.

      · Store HeLa Whole Cell Extract at -80°C. Avoid repeated freeze-thaw cycles. Stable at -80°C for 6 months.

      · Store the NFkB Capture Probe, Competitor Oligonucleotides, and Negative Control Probe at -20°C. May be stored at 4°C for less than one month.

      · Store the Transcription Factor Assay (TFA) Buffer at -20°C. Avoid freeze-thawing of the TFA Buffer. Buffer can be aliquotted.

      · Store all other components at 4°C.
      Packaging Information
      Material Size1 plate
      Material Package96 wells
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      NFκB p50/p65 EZ-TFA Transcription Factor Assay (Colorimetric) SDS

      Title

      Safety Data Sheet (SDS) 

      NFκB p50/p65 EZ-TFA Transcription Factor Assay (Colorimetric) Certificates of Analysis

      TitleLot Number
      NFkB p50/p65 EZ-TFA Transcription Factor Assay Colorimetric (500 series) and Chemiluminescent (600 series) Systems - 20141842014184
      NFkB p50/p65 EZ-TFA Transcription Factor Assay Colorimetric (500 series) and Chemiluminescent (600 series) Systems - 20355652035565
      NFkB p50/p65 EZ-TFA Transcription Factor Assay Colorimetric (600 series) and Chemiluminescent (600 series) Systems2941772
      NFkappaB p50/p65 EZ-TFA Transcription Factor Assay (Colorimetric) - anylotanylot
      NFκB p50/p65 EZ-TFA Transcription Factor Assay2453276
      NFκB p50/p65 EZ-TFA Transcription Factor Assay Colorimetric (500 series) and Chemiluminescent (600 series) Systems - 23910862391086
      NFκB p50/p65 EZ-TFA Transcription Factor Assay -26648972664897
      NFκB p50/p65 EZ-TFA Transcription Factor Assay -26730802673080
      NFκB p50/p65 EZ-TFA Transcription Factor Assay -26793882679388
      NFκB p50/p65 EZ-TFA Transcription Factor Assay Colorimetric (500 series) and Chemiluminescent (600 series) Systems3028340

      References

      Reference overviewPub Med ID
      Inhibition of hyaluronan synthesis reduced inflammatory response in mouse synovial fibroblasts subjected to collagen-induced arthritis.
      Giuseppe M Campo,Angela Avenoso,Angela D'Ascola,Vera Prestipino,Michele Scuruchi,Giancarlo Nastasi,Alberto Calatroni,Salvatore Campo
      Archives of biochemistry and biophysics  518  2012

      Show Abstract
      22197458 22197458
      Missing pieces in the NF-kappaB puzzle
      Ghosh, Sankar and Karin, Michael
      Cell, 109 Suppl:S81-96 (2002)  2002

      11983155 11983155
      Postrepression activation of NF-kappaB requires the amino-terminal nuclear export signal specific to IkappaBalpha
      Huang, T T and Miyamoto, S
      Mol Cell Biol, 21:4737-4747 (2001)  2001

      11416149 11416149

      Brochure

      Title
      Pathways and Biomarkers of Toll-like Receptor (TLR) Signaling (EMD)