NF-kappa B Activation Assay Kit | Syn: NF-kB, NF-{kappa}B, Nuclear Factor-Kappa B | Part NFKB-2

NF-kappa B Activation Assay Kit | Syn: NF-kB, NF-{kappa}B, Nuclear Factor-Kappa B | Part NFKB-2
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Price: $375.00
Availability: In Stock
Model: NFKB-2
Manufacturer: FIVEphoton Biochemicals

NF-kappa B Activation Assay Kit
 
Highlights of the NF-kB (NF-{kappa}B, Nuclear Factor-kappa Beta) Assay Kit 
  • Measure NF-kappa B activation in a wide range of mammalian species (human, mouse, rat, hamster, bovine, equine, porcine, canine) using cells or tissues with simple and rapid methodology. 
  • Suitable for 50 to 150 NF-kappa B activation assays based on one assay per semi-confluent 10 cm cell culture dish, or a well in a 6-well culture dish, respectively.
  • The NF-kappa B (NF-kB) assay kit offers a more reliable, selective and specific quantitative analysis compared to ELISA formats.  To detect NF-kappa B (NF-kB) activation, cells are partitioned into nuclear and cytoplasmic fractions in approximately 45 min using the provided reagents.  The ratio of p65(RelA) in the nuclear relative to the cytoplasmic fraction is quantitated in Western blots that are developed with primary and secondary antibodies that are provided with the kit.
  • Independently validated in published investigations.

   NF-kB (NF-kappa B) Activation Assay, Background  

Background

Following stimuli that elicit the NF-kB pathway, the IkB inhibitory subunit of the NF-kappa B complex is phosphorylated, ubiquitinated and degraded, exposing nuclear targeting signals in the transcription factor subunits of the NF-kappa B complex.  The NF-kappa B transcription factor subunits, p65 and p50, with exposed nuclear targeting signals then translocate into the nucleus from the cytoplasm.  This NF-kappa B assay kit provides an accurate quantitative estimate of NF-kappaB activation by measuring p65 (RelA) protein that has translocated to the nucleus.
 
Protocol Synopsis

In brief, the NF-kB assay isolates nuclear and cytoplasmic protein fractions, with concentrated p65(RelA) protein partitioned in the nuclear fraction:  The fractionation steps are completed in approximately 45 minutes.  Western blotting, using provided antibodies, is then used to detect and quantitate cytoplasmic and nuclear p65(RelA).  To separate cytoplasmic from nuclear fraction, 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 washed in the Cytoplasmic Fractionation Reagent and then extracted with the "Nuclear Fractionation Reagent (Part No. NER-1)."  The nuclear - cytoplasmic fractionation steps are conveniently completed in approximately 45 min using snap-cap tubes and a table-top centrifuge.  Aliquots of each fraction are then resolved in gels and Western blots, with consecutive lanes loaded to display total cell lysate, cytoplasmic fraction and nuclear fraction.  The ratio of p65(RelA) protein in the nuclear relative to the cytoplasmic fraction is then calculated.    This ratio provides a quantitative measurement of p65 translocation and an index of NF-kappaB activation.

 



 Kit Contents
   Part No.
  Description
  Volume
1.
    CER-1
  Cytoplasmic Fractionation Reagent
  55 ml
2.
    NER-1
  Nuclear Fractionation Reagent
  3.5 ml
3.
    DTT
  Dithiothreitol
  (solid)
4.
    p65Ab
  Rabbit IgG antibody to p65(RelA)
  100 µl
5.
   G-HRP
  Goat anti-rabbit IgG-HRP
  50 µl

Antibody to p65(RelA) for Western Blots (provided with the kit)

Antibody to p65(RelA) for Western blot is provided in 100 microliter aliquots (rabbit polyclonal, IgG; applicable at 1:400 dilution).  The antibody is reactive toward human, mouse, hamster, rat, bovine, porcine, murine, equine, and canine p65.  A matched goat- anti-rabbit IgG-HRP antibody is also included in the kit for chemiluminescent Western blot detection.


NFKB-2 Kit Data

Data revealing nuclear and cytoplasmic fractionation, NF-kappa B activation and reactivity of the provided p65 antibody.  All data derived from transfected HEK293 cells. The images below can be enlarged by right clicking and selecting view image (Mac: Ctl click, select view image).
 

                                                   A                                                                          B                                                                             C
 
Figure A:  Western blot and quantitative analysis revealing NF-kappa B activation over three experimental repetitions elicited by overexpression of SNP variants of a gene associated with a neurological disease.  Lane 1 in the Western blot and the corresponding bar chart reflect the mock transfection control (nl).  Lane 2 in the Western blot and the corresponding bar chart reveal mutant variant SNP-1 expression (S1).  Lane 3 in the Western blot and bar chart reveal the NF-kappa B response to SNP-2 (S2) expression.  Western blot data display elevated levels of p65 subunit protein in the nuclear fraction corresponding to expression of SNPs 1 and 2, implicating cellular stress and NF-kappa B activation.  Figure B:  An independent, identical experiment to that displayed in Figure 1, revealing elevated translocation of p65 into the nuclear fraction following transfection of SNP variants 1 to 3 of the gene associated with a neurological disease.  Comparisons were done with p65 protein levels in nuclear fractions relative to p65 levels in total cell lysate.  Transfection of SNP-3 increases p65 protein levels in the nuclear fraction, indicating elevated NF-kappa B activation. Figure C Activation of NF-kappa B after expression of a trafficking impaired transmembrane protein (Tmp; view total cell Lysate) and cellular stress revealed by elevated nuclear p65 protein levels (Nuc).  M: mutant protein. wt: wild type protein.  Cyt:  cytoplasmic fraction. 

                                                                    


Product Citations

  1. Thorsten R Doeppner, Britta Kaltwasser, Jin Fengyan, Dirk M Hermann and Mathias Bähr.  2013.  TAT-Hsp70 induces neuroprotection against stroke via anti-inflammatory actions providing appropriate cellular microenvironment for transplantation of neural precursor cells.  Journal of Cerebral Blood Flow & Metabolism 33, 1778-1788 (November 2013).  Link to article
  2. Musa Mulongo et. al.  2013.  In vitro infection of bovine monocytes with Mycoplasma bovis delays apoptosis and suppresses production of IFN-γ and TNF-α but not IL-10. 
  3. Huang Z, Rose AH, Hoffmann FW, Hashimoto AS, Bertino P, Denk T, Takano J, Iwata N, Saido TC, Hoffmann PR.  2013
    Calpastatin Prevents NF-κB–Mediated Hyperactivation of Macrophages and Attenuates ColitisJ. Immunol.  2013 Oct 1;191(7):3778-88. doi: 10.4049/jimmunol.1300972. Epub 2013 Aug 28.  Link to article
  4. Amelia A. Romoser et al.  2011.  Quantum dots trigger immunomodulation of the NFκB pathway in human skin cells.  Mol Immunol. 2011 July; 48(12-13): 1349–1359.  Link to article

References on NF-KB Signaling and Activation Assays
  1. Baltimore D.  Discovering NF-kappaB. 2009  Cold Spring Harb Perspect Biol. Jul;1(1): a000026.
  2. Bellarosa D, Binaschi M, Maggi CA, Goso C. 2005. Sabarubicin- (MEN 10755) and paclitaxel show different kinetics in nuclear factor-kappaB (NF-kB) activation: effect of parthenolide on their cytotoxicity. Anticancer Res 25: 2119-28.
  3. Campo GM, Avenoso A, Campo S, D'Ascola A, Traina P, Calatroni A. 2008. Chondroitin-4-sulphate inhibits NF-kB translocation and caspase activation in collagen-induced arthritis in mice. Osteoarthritis Cartilage 16: 1474-83.
  4. Cascinu S, Berardi R, Salvagni S, Beretta GD, Catalano V, et al. 2008. A combination of gefitinib and FOLFOX-4 as first-line treatment in advanced colorectal cancer patients. A GISCAD multicentre phase II study including a biological analysis of EGFR overexpression, amplification and NF-kB activation. Br J Cancer 98: 71-6.
  5. Chen HH, Chen TW, Lin H. 2009. Prostacyclin-induced peroxisome proliferator-activated receptor-alpha translocation attenuates NF-kappaB and TNFalpha activation after renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 297: F1109-18.
  6. Ea CK, Baltimore D.  Regulation of NF-kappaB activity through lysine monomethylation of p65. 2009. Proc Natl Acad Sci USA.  106(45):18972-7
  7. Harder J, Franchi L, Munoz-Planillo R, Park JH, Reimer T, Nunez G. 2009. Activation of the Nlrp3 inflammasome by Streptococcus pyogenes requires streptolysin O and NF-kappa B activation but proceeds independently of TLR signaling and P2X7 receptor. J Immunol 183: 5823-9.
  8. Kaiser WJ, Upton JW, Mocarski ES. 2008. Receptor-interacting protein homotypic interaction motif-dependent control of NF-kappa B activation via the DNA-dependent activator of IFN regulatory factors. J Immunol 181: 6427-34.
  9. Kanda T, Yokosuka O, Nagao K, Saisho H. 2006. State of hepatitis C viral replication enhances activation of NF-kB- and AP-1-signaling induced by hepatitis B virus X.Cancer Lett 234: 143-8.
  10. Karin M.  NF-kappaB as a critical link between inflammation and cancer. 2009  Cold Spring Harb Perspect Biol.  a000141. Review
  11. Karin, M. and Greten, F. R. 2005. NF-kappaB:  linking inflammation and immunity of cancer development and progression.  Nat Rev Immunol. 5 (10), 749-759.
  12. Kingeter LM, Schaefer BC. 2008. Loss of protein kinase C theta, Bcl10, or Malt1 selectively impairs proliferation and NF-kappa B activation in the CD4+ T cell subset. J Immunol 181: 6244-54.
  13. Lim EJ, Lee SH, Lee JG, Kim JR, Yun SS, et al. 2007. Toll-like receptor 9 dependent activation of MAPK and NF-kB is required for the CpG ODN-induced matrix metalloproteinase-9 expression. Exp Mol Med 39: 239-45.
  14. Mefert MK, Baltimore D.  2005.  Physiological functions of brain NF-kappaB. Trends Neurosci.  28(1): 37-43.
  15. Nicola JP, Nazar M, Mascanfroni ID, Pellizas CG, Masini-Repiso AM. NF-{kappa}B p65 Subunit Mediates Lipopolysaccharide-Induced Na+/I- Symporter Gene Expression by Involving Functional Interaction with the Paired Domain Transcription Factor Pax8. Mol Endocrinol.
Version 9-30-2014

Safety, Storage, Shipping, NF-kappa B Activation Assay Kit, Part No. NFKB-2 
Safety:  Irritant.  Avoid ingestion, eye and skin contact.
Storage:  Store antibodies at 4oC.  Other contents at -20oC.
Recommended Shipping.  Ships at ambient temperature.  Domestic overnight and 2-day delivery and international priority delivery is available,  Fully stable for international shipping to Canada, Europe, Asia, Middle East, and South America at ambient temperature.



 

Category Download Link
Protocol Manual click here
p65 Antibody Data Sheet
click here
MSDS click here