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70-610 NFκB p50/p65 EZ-TFA Transcription Factor Assay (Chemiluminescent)

1 plate  96 wells
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      Key Specifications Table

      Key Applications
      Catalogue Number70-610
      Brand Family Upstate
      Trade Name
      • EZ-TFA
      • Upstate
      DescriptionNFκB p50/p65 EZ-TFA Transcription Factor Assay (Chemiluminescent)
      OverviewThe Non-Radioactive Chemiluminescent NFκB 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 flanked DNA binding consensus sequence for NFκB (5'-GGGACTTTCC-3'), is mixed with cellular (nuclear) extract in the Transcription Factor Assay Buffer provided. When incubated together, the active form of NFκB contained in the nuclear extract binds to its consensus sequence. The extract/probe/buffer mixture is then directly transferred to the streptavidin-coated plate. The active NFκB protein is immobilized on the biotinylated double stranded oligonucleotide capture probe bound to the streptavidin plate well, and any inactive, unbound material is washed away. The bound NFκB transcription factor subunits, p50 and/or p65, are detected with specific primary antibodies, a Rabbit anti-NFκB p50 and a Rabbit anti-NFκB p65. A highly sensitive HRP-conjugated secondary antibody is then used for detection. This provides sensitive chemiluminescent detection that can be read in a microplate luminometer or by a CCD camera-coupled imaging system. Included in the kit are positive cell extract, a non-specific double stranded oligonucleotide, and a specific competitor double stranded oligonucleotide. The NFκB Transcription Factor Assay was QC tested using nuclear extracts from human (HeLa) cells. Due to the conservation in the NFκB DNA binding site and the fact that the primary antibodies contained in this kit cross-react with rat and mouse NFκB, this assay is expected to work with samples from rat and mouse as well as human.
      Background InformationThe transcription factor NF&jkappa;B (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 repeats1,2. NFκB 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, NFκB1 (p50 and its precursor p105), and NFκB2 (p52 and its precursor p100)1, 2. NFκB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes. Although most NFκB 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 NFκB is the most abundant in cells3. A critical component in NFκB regulation is the IκB Kinase (IKK) complex. In a majority of unstimulated cells, the NFκB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IκB proteins4,5. Upon stimulation by multiple inducers including viruses or cytokines, such as TNFα, IL-1, or PMA, I&kappaBα is rapidly phosphorylated and degraded, resulting in the release of the NFκB 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. NFκB induces the transcription of its own inhibitor, IκB&alapha;, causing an autoregulatory mechanism of NFκB activity and generating the inactive form of NFκB. The newly formed nuclear NFκB-IκBα complexes are then exported out to the cytoplasm, thereby reestablishing the cytoplasmic pool of inactive NFκB complexes primed for another round of activation to take place2. The wide variety of genes regulated by NFκB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins, and inducible effector enzymes3.
      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)
      Product Information
      • Primary Antibody (Part No. 90274): One vial containing 15 μL Rabbit anti-NFκB p65. Use at 1:1000 dilution in Enhanced Transcription Factor Assay Buffer (see 70-600 Universal Transcription Factor Assay product manual).
      • Primary Antibody (Part No. 90275): One vial containing 15 μL of anti-NFκB p50. Use at 1:1000 dilution (can be used at 1:500 dilution when looking at μg quantities of nuclear extract to give a higher, more sensitive signal) in Enhanced Transcription Factor Assay Buffer (see Assay Protocol).
      • NFκB Capture Probe (Part No. 90269): One vial containing 220 μL biotinylated double stranded oligonucleotide probe (1 pmol/μL) containing the flanked wild-type consensus sequence (5'-GGGACTTTCC-3') for NFκB.
      • NFκB Specific Competitor Oligonucleotide (Part No. 90270): One vial containing 20 μL (10 pmol/μL) specific competitor oligonucleotide. The NFκB Competitor Oligonucleotide has the same consensus sequence
      • TFA Negative Control Probe (Part No. 90271): One vial containing 20 μL biotinylated double stranded oligonucleotide probe (2 pmol/μL). Provided as an internal negative control, this non-specific competitor oligonucleotide does not contain the STAT consensus sequences.
      • HeLa Whole Cell Extract, TNFα Treated (Part No. 90350): One vial containing 25 μL TNFα stimulated HeLa whole cell extract. This extract is provided as a positive control and is not intended for extract-to-extract, nor plate-to-plate comparisons. When using this control, you will notice a decrease in signal with repeated freeze/thaw cycles. This component must be stored at -80°C.
      • EZ-TFA (Universal Transcription Factor Assay, Chemiluminescent) Catalog #70-600: One universal kit containing 80 mL TFA Buffer, one streptavidin-coated stripwell plate, 12 grams of blocking reagent, 250 μl 10X annealing buffer, 12 mL TMB/E, 12 mL stop solution, 30 μl Gt X Rb HRP secondary antibody, 30 μl Gt X Ms HRP secondary antibody. A detailed manual is also included with the 70-600 Universal TFA component. The procedures described in the manual should be consulted as well as the specific product information in this 70-610/615/620 product specific manual.
      Quality LevelMQ100
      ApplicationThe Non-Radioactive Chemiluminescent NFκB p50/p65 Transcription Factor Assay kit includes a positive cell extract, a non-specific double stranded oligonucleotide & a specific competitor double stranded oligonucleotide.
      Key Applications
      • Affinity Binding Assay
      • ELISA
      • Activity Assay
      Biological Information
      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
      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


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

      TitleLot Number
      NFkappaB p50/p65 EZ-TFA Transcription Factor Assay (Chemiluminescent) - anylotanylot
      NFkappaB p50/p65 EZ-TFA Transcription Factor Assay (Chemiluminescent) - anylotanylot
      NFκB p50/p65 EZ-TFA Transcription Factor Assay -26648972664897
      NFκB p50/p65 EZ-TFA Transcription Factor Assay -26730802673080


      Reference overviewPub Med ID
      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


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