NF-kappa B, NF-{kappa}B, NF-kB, Nuclear Factor-Kappa B, Activation Assay Kit (Part No. NFKB-1)

NF-kappa B, NF-{kappa}B, NF-kB, Nuclear Factor-Kappa B, Activation Assay Kit (Part No. NFKB-1)
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Price: $179.95
Availability: In Stock
Model: NFKB-1
Manufacturer: FIVEphoton Biochemicals

NF-kappa B, (NF-{kappa}B, NF-KB) Activation Assay Kit

 

 

 

Highlights the NF-kappa B Activation Assay Kit, Part No. NFKB-1

  • Rapid assay to detect and quantitate NF-kappa B activation by isolating nuclear and cytoplasmic fractions and detection of NF-KB transcription factor subunit (i.e. p65 and p50) enrichment in the nuclear fraction by Western blot.
  • Nuclear and cytoplasmic fractions are isolated in less than 1 hour using a microcentrifuge.
  • Data from this NF-  K  B Activation Assay Kit are higher resolved, easier to quantitate and more convincing than fluorescence and luminescence assays, including ELISA.
  • The NF-KB Activation Assay Kit is applicable in virtually all mammalian cells.
  • For 50 to 150 NF-kappa B activation assays based on one assay per semi-confluent 10 cm cell culture dish or well of a 6-well dish, respectively.

View Protocol


NF-kappa B Activation Assay Kit:  Overview

 

        
  
  The NF-kappa B Activation Assay Kit contains pre-configured reagents to assay NF-kappa B activation by separating cells into nuclear and cytoplasmic fractions, and by detecting transcription factor enrichment in the nuclear fraction.  Cell fractionation into cytoplasmic and nuclear fractions is conveniently performed in 1.5 ml tubes and a microcentrifuge in approximately 45 minutes.            Nuclear proteins, including activated transcription factor subunits of the NF-kappa B complex, such as p65, are enriched in the nuclear fraction.         The transcription factor subunits of the NF-kappa B complex in the nuclear and cytoplasmic fractions are detected and quantitated with Western blot analysis.
 

 

 

 

   

 

 

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." The cell suspension is briefly centrifuged in a microcentrifuge, which generates the cytoplasmic fraction in the supernatant.  The pellet is then washed in the Cytoplasmic Fractionation Reagent and extracted with the "Nuclear Fractionation Reagent."  Aliquots of each fraction are resolved in Western blots with consecutive lanes loaded to display total cell lysate, cytoplasmic fraction and nuclear fraction.  The extent of translocation from the cytoplasm to nucleus reveals a quantitative assessment of NF-kappa B activation.
 
Icon Image:  Crystal structure of the p50-Rel subunit complex of NF-kappa B bound to HIV DNA:  Source, NCBI Protein Data Bank. 

 


Data:  NF-kappa B Activation Assay Kit (NFKB-1.  Enlargements can be viewed in the Images and Data tab).  All data presented below are from transfected HEK293 cells.

                              Fig. 1                                              Fig. 2                                                       Fig. 3                                                        

Figure Legends:  Representative data derived from the FIVEphoton Biochemicals NF-kappa B Activation Assay Kit in transfected HEK293 cells:  Fig. 1.  Western blot and quantitative analysis revealing NF-kappa B activation in three experimental repetitions elicited by transfection and overexpression in HEK293 cells of SNP variants of a gene associated with a neurological disease:  SNP overexpression activates a cellular stress response in HEK293 cells.  Lane 1 in the Western blot and corresponding bar chart reflect the mock transfection control (nl).  Lane 2 and the corresponding bar chart display the NF-kappa B response to expression of mutant variant SNP-1 (S1) of the gene.  Lane 3 in the Western blot and corresponding bar chart reveal the NF-kappa B response due to expression of SNP-2 (S2). Fig. 2:  Data from an independent experiment revealing elevated translocation of p65 into the nuclear fraction.  Fig. 3:  Activation of NF-kappa B and increased levels of p65 protein in the nuclear fraction following TNFalpha treatment of HEK293 cells. TNFalpha activates the NF-kappa B pathway.

 


NF-kappa B Activation Assay Kit Components

  1. Cytoplasmic Fractionation Reagent  (CER-1;  55 ml)
  2. Nuclear Fractionation Reagent (NER-1;  3.5 ml)
  3. DTT (sufficient to make a IM solution, volume 64 µl)

Related NF-KB Activation Assay Kits

The NFKB-2, NF-kappa B Activation Assay Kit - 2, provides reagents for cell fractionation as well as antibody to detect p65 in Western blots from many mammalian cells, including human, rodent, and canine.  Click button to view kit NFKB-2.        

            

 


 

Representative References on NF-kappa B Signaling    
  1.   Chen L, Yang G, Zhang X, Wu J, Gu Q, et al. 2009. Induction of MIF expression by oxidized LDL via activation of NF-kappaB in vascular smooth muscle   cells. Atherosclerosis 207: 428-33.Hoffmann, A. and Baltimore, D. (2006) Circuitry of nuclear factor kappaB signaling. Immunol Rev 210, 171-186.   
  2. Hoffmann F, .Sass G, Zillies J, Zahler S, Tiegs G, et al. 2009. A novel technique for selective NF-kappaB inhibition in Kupffer cells: contrary effects in fulminan hepatitis and ischaemia-reperfusion. Gut 58: 1670-8.
  3. Itoh M, Fu L, Tohda S. 2009. NF-kappaB activation induced by Notch ligand stimulation in acute myeloid leukemia cells. Oncol Rep 22: 631-4.
  4. Karin, M. (2006) Nuclear factor-kappaB in cancer development and progression. Nature 441 (7092), 431-436.
  5. Karin, M. and Greten, F.R. (2005) NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 5 (10), 749-759.
  6. Osterlund C, Gronlund H, Polovic N, Sundstrom S, Gafvelin G, Bucht A. 2009. The non-proteolytic house dust mite allergen Der p 2 induce NF-kappaB and MAPK dependent activation of bronchial epithelial cells. Clin Exp Allergy 39: 1199-208.
  7. Wirth, T. and Baltimore, D. (1988) Nuclear factor NF-kappa B can interact functionally with its cognate binding site to provide lymphoid-specific promoter function. Embo J 7 (10), 3109-3113.
  8. Wu S, Yin R, Ernest R, Li Y, Zhelyabovska O, et al. 2009. Liver X receptors are negative regulators of cardiac hypertrophy via suppressing NF-kappaB   signalling. Cardiovasc Res 84: 119-26.
  9. Zhang L, Altuwaijri S, Deng F, Chen L, Lal P, et al. 2009. NF-kappaB regulates androgen receptor expression and prostate cancer growth. Am J Pathol 175: 489-99

Storage:  NF-kappa B Activation Assay Kit:   Storage:  -20oC upon arrival.
 
Safety:  Irritant.  Use gloves and eye protection.  Avoid skin and eye contact.
Shipping:  NF-kappa B Activation Assay Kit:  Domestic 2-day (flat Fedex rate, $26) and overnight shipping (flate Fedex rate, $42).  International delivery is also available for NF-kappaB Activation Assay Kits;  please inquire with salessupport@fivephoton.com for rate.

 

 

 


 

Category Download Link
Protocol Manual click here
MSDS click here




NF-kappa B, NF-{kappa}B, NF-kB, Nuclear Factor-Kappa B, Activation Assay Kit (Part No. NFKB-1)
Click to enlarge
NF-kappa B, NF-{kappa}B, NF-kB, Nuclear Factor-Kappa B, Activation Assay Kit (Part No. NFKB-1)
Click to enlarge
NF-kappa B, NF-{kappa}B, NF-kB, Nuclear Factor-Kappa B, Activation Assay Kit (Part No. NFKB-1)
Click to enlarge