Rat RelA p65 NF-kappa B (NF-kB, Nuclear Factor-kappa B) ELISA Assay Kit (Part No. NFKB-ELISA (r, 96T))

Rat RelA (p65) ELISA Kit for NF-kB

Lead time:  Approximately 2 weeks

Email customersupport@fivephoton.com for current lead time estimate.

• Measures total RelA(p65) in sandwich ELISA format
  
• Affinity purified polyclonal capture antibody with polyclonal HRP-conjugated detection antibody
  
• Strip plates
  
• p65 Standard Concentration:  2700pg/ml 
• Assay Range:  30pg/ml - 1800pg/ml
• HRP-colorimetric detection
• Rapid Assay:  Total incubation time with plate and HRP-detection antibody 1hr at 37^oC
• Nuclear-cytoplasmic isolation kit is offered to quantify nuclear p65 (see available options menu).           

View Protocol

---

Background

The RelA p65 NF-kB (NF-kappa B, NF-{kappa}B) ELISA kit employs the sandwich ELISA(96T) format to quantitate NF-kB p65 protein levels.  The 96-well strip plate is coated with an affinity purified polyclonal capture antibody selective to the RelA (p65) subunit of NF-kB.  After addition of sample, incubation and wash, a second polyclonal anti-RelA (p65) HRP-conjugated antibody is added to each well.  Color reagents are added to generate a colorimetric reaction, which corresponds to the concentration of RelA (p65) in the sample.

A Nuclear-Cytoplasmic fractionation kit is offered to partition cytoplasmic and nuclear p65.  p65(RelA) in the nuclear and cytoplosmic fractions can be directly measured with this kit to provide an estimate of NF-kappa B activation by calculation of the nuclear p65 relative to cytoplasmic p65 concentration ratio.

---

Solid Support Sandwich ELISA to RelA p65 NF-kB (NF-kappa B) Methods

 

Steps of Sandwich ELISA
1.  Equilibrate plate to ambient temperature.

2.  Add sample, incubate 30 min - 1hr, wash.

3.  Add HRP-Antibody, incubate 30 min -1 hr, wash.

4.  Add color reagents, incubate 15 min.

5.  Add stop solution.  Read Absorbance 450 nm.

---

References of RelA p65 NF-kB (NF-kappaB) Assays, Including ELISA

1. Baudy, A.R. et al. (2009) A robust in vitro screening assay to identify NF-kappaB inhibitors for inflammatory muscle diseases. Int Immunopharmacol 9 (10), 1209-1214.
   
2. Dann, S.G. et al. (2004) Chromatin immunoprecipitation assay on the rainbow trout opsin proximal promoters illustrates binding of NF-kappaB and c-jun to the SWS1 promoter in the retina. Exp Eye Res 78 (5), 1015-1024.
   
3. Hassan, N.J. et al. (2009) Development of an insect-cell-based assay for detection of kinase inhibition using NF-kappaB-inducing kinase as a paradigm. Biochem J 419 (1), 65-73.
   
4. Hellweg, C.E. et al. (2003) Generation of stably transfected Mammalian cell lines as fluorescent screening assay for NF-kappaB activation-dependent gene expression. J Biomol Screen 8 (5), 511-521.
   
5. Jin, S. et al. (2005) A simplified probe preparation for ELISA-based NF-kappaB activity assay. J Biochem Biophys Methods 65 (1), 20-29.
   
6. Kaur, J. et al. (2008) Chemopreventive activity of lantadenes on two-stage carcinogenesis model in Swiss albino mice: AP-1 (c-jun), NFkappaB (p65) and P53 expression by ELISA and immunohistochemical localization. Mol Cell Biochem 314 (1-2), 1-8.
   
7. Klinke, D.J., 2nd et al. (2008) Modulating temporal control of NF-kappaB activation: implications for therapeutic and assay selection. Biophys J 94 (11), 4249-4259.
   
8. Li, J.L. et al. (2007) An optimized assay for transcription factor NF-kappaB with dsDNA-coupled microplate. Colloids Surf B Biointerfaces 55 (1), 31-37.
   
9. Moon, K.Y. et al. (2001) A cell-based assay system for monitoring NF-kappaB activity in human HaCat transfectant cells. Anal Biochem 292 (1), 17-21.
   
10. Noursadeghi, M. et al. (2008) Quantitative imaging assay for NF-kappaB nuclear translocation in primary human macrophages. J Immunol Methods 329 (1-2), 194-200.
    
11. Sun, C.S. et al. (2008) Anti-sense morpholino oligonucleotide assay shows critical involvement for NF-kappaB activation in the production of Wnt-1 protein by HepG2 cells: oncology implications. J Biomed Sci 15 (5), 633-643.
    
12. Tomita, K. et al. (1999) Functional assay of NF-kappaB translocation into nuclei by laser scanning cytometry: inhibitory effect by dexamethasone or theophylline. Naunyn Schmiedebergs Arch Pharmacol 359 (4), 249-255.
    
13. Wang, J. et al. (2006) Exonuclease-mediated ELISA-like assay for detecting DNA-binding activity of transcription factors: measurement of activated NF-kappaB. Biotechniques 41 (1), 79-88, 90.
    

---

RelA p65 NF-kB (NF-{kappa}B, NF-kappaB, Nuclear Factor-KappaB) ELISA Kit

Safety:  Stop solution contains sulfuric acid.  Avoid all contact and inhalation.
Wear eye protection, gloves.

Storage:  4^oC, 6 months.

Shipping:  Overnight domestic shipping with ice pack is recommended for the NF-kB ELISA Kit (Cost:  $52.00).