NF-kappa B (NF-KB) - p65 (RelA) Immunofluorescence Labeling Kit (NF-kappaB, NFKB-3)

NF-kappa B (NF-KB) - p65 (RelA)  Immunofluorescence Labeling Kit (NF-kappaB, NFKB-3)
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Price: $395.00
Availability: In Stock
Model: NFKB-3
Manufacturer: Fivephoton Biochemicals

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NF-kappa B - p65 (NF-kB, Nuclear Factor-kappa B, RelA) Immunofluorescence Translocation Labeling Kit

Highlights of Kit NFKB-3
  • Kit includes matched primary and 2o fluorophore-tagged (rhodamine, Fab fragment) antibodies to detect p65 in nucleus and cytoplasm.
  • Applicable for most mammalian cells (e.g. human, mouse, rat, hamster, canine, rabbit, equine).
  • Provides a visual demonstration of NF-kappa B activation and p65 translocation.
  • NF-kB activation is quantified by measuring immunofluorescence intensity corresponding to p65 in cytoplasm and nucleus using pixel quantification software.*
  • Sufficient solutions are provided to label p65 in nucleus and cytoplasm in up to 40 microscope slides.
In addition to direct purchase from Fivephoton Biochemicals, this product can be purchased on fishersci.com and shipped directly from Fivephoton Biochemical to the customer;  Fishersci.com part number NC0314660.  The NF-kappa B Immunofluorescence Labeling Kit is also available from additional distributors of Fivephoton Biochemicals.
 
 
  
Additional methods to detect p65 transloation:  Trask OJ Jr. Nuclear Factor Kappa B (NF-κB) Translocation Assay Development and Validation for High Content Screening. 2012 Oct 1. In: Sittampalam GS, Coussens NP, Nelson H, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Available from: http://www.ncbi.nlm.nih.gov/books/NBK100914/
 
 

Background

The NF-kB complex resides in the cytoplasm in its inactive state consisting of the inhibitory IkB subunit assembled with the p65-p50 heterodimer through ankyrin repeats.  With stimulus, the IkB inhibitory subunit is ubiquitinated and degraded, exposing nuclear localization signals on the p65 (RelA) subunit, which then translocates into the nucleus.  The antibodies provided with this kit are employed to fluorescently label p65 in nucleus and cytoplasm.  Fluorescent microscopy and pixel quantitation software are then employed to estimate the emission intensity of p65 in the nucleus relative to the cytoplasm.

 
Methods Overview
 
Cells are grown on glass cover slips to approximately 85% confluency and treated to activate NF-kappa B.  Cellular proteins are first cross-linked and immobilized with paraformaldehyde (not provided).  The cross-linking reaction is quenched with a provided quenching solution.  Plasma and nuclear membranes are permeabilized with a provided solution that contains a permeabilization reagent and protein blockers.  An included anti-p65 IgG antibody is then applied to cells for 1hr.  Unbound antibody is washed away and a provided rhodamine tagged Fab fragment secondary antibody is added for an additional 1 hr.   Unbound secondary antibody is washed away, and a sandwich consisting of slide and cover slip, with antifade solution in-between, is configured.  The antifade solution is allowed to solidify overnight.  The microscope slide-cover slip configuration is now ready for visual and quantitative detection of p65 translocation.

 
Kit contents
 
Item
Description
Volume
1
PBS (10X)
16 ml
2
Permeabilization Buffer (10X)
8 ml
3
Quenching Buffer (10X)
25 ml
4
Intracellular Detection Buffer (10X)
60 ml
5
Polyclonal rabbit IgG to p65
120 µl
6
Anti-rabbit IgG Rhodamine (Fab fragment)
50 µl
7
Forceps set

 

Pixel Quantitation Software

Safety, Storage, Shipping NF-kappa B - p65 Immunofluorescence Kit
Safety:  Irritant.  Avoid ingestion, skin and eye contact
Storage:  Antibodies,  4oC:  Solutions, -20oC
Shipping:  Domestic 2-day Fedex;  $28:  Domestic Overnight Fedex; $42.  International Fedex priority shipping is also available.  Shipped at ambient temperature.  Store as recommended on the label after arrival.  Please inquire with salessupport@fivephoton.com for rate.
kw.  p65 rela translocation assay kit, nf-kappa b activation assay kit, nfkb, translocation assay, immunofluorescence assay
 



 

Category Download Link
Protocol Manual click here
p65 Antibody Sheet
click here
Anti-rabbit IgG-Rhodamine Sheet
click here
MSDS click here