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 Labeling Kit

Highlights of Kit NFKB-3
  • Kit includes matched primary and secondary fluorophore-tagged (rhodamine) antibodies to detect p65 in nucleus and cytoplasm.
  • Applicable to most mammalian cells (e.g. human, mouse, rat, hamster, canine, rabbit, equine).
  • Provides a visual demonstration of p65 translocation and NF-kappa B activation.
  • 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.
 

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.  Upon stimuli, 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 used to estimate the relative  emission intensity corresponding to 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.  To commence immunofluorescent labeling, cellular proteins are cross-linked and immobilized with paraformaldehyde.  The cross-linking reaction is quenched with a provided quenching solution.  Plasma and nuclear membranes are permeabilized with another provided solution that contains protein blockers to inhibit non-specific antibody binding.   The provided anti-p65 IgG antibody is applied to cells for one hour.  Unbound antibody is washed away and an included rhodamine tagged secondary antibody is added for another 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
50 µl
 

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.
 



 

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