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InformationHuman Cell NF-kappa B (NF-kB) Activation Assay Kit (Part No. hNFKB-4)
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NF-kappa B (NF-KB) Activation Assay Kit for Human Cells
For 50 - 150 NF-kappa B activation assays based on one assay per confluent 10 cm cell culture dish, or 6-well culture dishes, respectively. Includes selective and high affinity polyclonal rabbit IgG antibody reactive to human p65 (RelA) in Western blots.
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Rapid assay to quantitate NF-kappa B activation using cell fractionation into nuclear and cytoplasmic components
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Separation into nuclear and cytoplasmic components is complete in less than 45 minutes using a microcentrifuge
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Assay format provides a more specific quantitation of NF-kappa B activation compared to ELISA format
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Data and Figure Legends are presented in Images and Data tab from transfected HEK cells
Principles of the Human NF-kappa B Activation Assay Kit
This NF-KB activation assay follows the translocation of the p65 (RelA) subunit of NF-kB into the nucleus, a hallmark of NF-kappa B activation. Cytoplasmic and nuclear fractions are isolated, and Western blots of the fractions are used to quantify the relative p65 concentration. The ratio of nuclear relative to cytoplasmic p65 concentration provides a numerical estimate of NF-kappa B activation.
This NF-KB Activation Assay Kit provides all the reagents required to fractionate cells into cytoplasmic and nuclear fractions, as well as a selective and high affinity antibody to detect p65 in Western blots.
Kit Contents:
- Cytoplasmic Fractionation Reagent (CER-1; 55 ml)
- Nuclear Fractionation Reagent (NER-1; 3.5 ml)
- DTT (sufficient to make a 1M solution, volume 64 microliters)
- Rabbit Polyclonal IgG Antibody to p65. 100 microliters provided: Use at 1:100 - 1:400 dilution for Western blots
NF-kappa B Activation Assay; General Protocol
To separate cytoplasmic from nuclear fraction following NF-kappa B activation, cells are first exposed to the "Cytoplasmic Fractionation Reagent (Part CER-1)." The cell suspension is briefly centrifuged in a microcentrifuge, which generates the cytoplasmic fraction in the supernatant. A resulting pellet is then washed in the Cytoplasmic Fractionation Reagent and extracted with the "Nuclear Fractionation Reagent (Part No. NER-1)." Aliquots of each fraction are resolved in Western blots, with consecutive lanes loaded to display total cell lysate, cytoplasmic fraction and nuclear fraction. The Western blots are developed with the provided anti-p65 antibody. Western blot band densities of p65 in cytoplasmic and nuclear fractions are measured, and the ratio of nuclear relative to cytoplasmic p65 is calculated: this ratio provides a numerical estimate of NF-kappa B activation.
Icon Image: Crystal structure of the p50-p65 subunit complex of NF-kappa B bound to HIV DNA. Source: NCBI Protein Data Bank.
Product Citations
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Dattaroy, D et. al.2015. Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis.Vol.no. DOI:
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, X-Y, et. al. 2014. Glyoxalase I Is Differentially Expressed in Cutaneous Neoplasms and Contributes to the Progression of Squamous Cell Carcinoma, Journal of Investigative Dermatology (3 September 2014) | doi:10.1038/jid.2014.377. Link to article
Human NF-kappa B Activation Assay Kit, Safety, Storage and Shipping
Safety: Contains irritants. Avoid ingestion, eye and skin contact
Storage: p65 Antibody: Aliquot, -20oC: 4oC. Other contents: -20oC
Shipping: Ships at ambient temperature. Fedex 2-day domestic delivery: $27. Fedex domestic overnight delivery: $45. International Fedex priority Fedex shipping: Estimated $85 (Asia and Western Europe)-$120 (Scandanavia), depending on location. Please inquire with salessupport@fivephoton.com for details on international shipping rates.
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 ELISA Kit - FIVEphoton Biochemicals copy-80x80.jpg "Mouse RelA p65 NF-kappa B (NF-kB, Nuclear Factor-kappa B) ELISA Assay Kit. Part No. ELISA-NFKB(m, 96T)")
