p65(RelA) Translocation Assay Kit (Part No. p65-Trans)

p65(RelA) Translocation Assay Kit

• Rapid, quantitative assay to measure translocation of p65 from cytoplasm to nucleus.
  
• Generates a nuclear fraction with concentrated p65 protein.
• This assay operates in most mammalian cell types. 
• Data displayed in Images and Data tab

View Protocol

Data displaying p65(RelA) translocation;  click "Images and Data" tab

Figure Legends:  All data derived from transfected HEK293 cells.  First Figure:  Western blot and quantitative analysis revealing NF-kappa B activation and p65 translocation as determined from three experimental repetitions elicited by transfection and 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 expression of the mutant variant SNP-1 (S1) of the gene.  Lane 3 in the Western blot and the corresponding bar chart reveal the p65 translocation response to expression of SNP-2 (S2).  Western blot data display elevated levels of p65 subunit protein in the nuclear fraction corresponding to expression of SNPs 1 and 2, demonstrating cellular stress and NF-kappa B activation.  Second Figure:  In an independent and repetitive experiment to that displayed in Figure 1, data 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 (relative to expression of the wild-type variant;  WT).  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 and cellular stress.  Third Figure: Activation of NF-kappa B and p65 translocation after expression of a trafficking impaired transmembrane protein (Tmp; displayed in total cell Lysate) and activation of endoplasmic reticulum stress as revealed by elevated p65 protein levels in the nuclear fraction (Nuc).  Legend: M: mutant protein.  wt: wild type protein.  Cyt: cytoplasmic fraction.  Nuc: nuclear fraction.   

The assay isolates a nuclear fraction with concentrated p65, as well as a cytoplasmic fraction.  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)."  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 p65 translocation from the cytoplasm into the nucleus provides a quantitative estimate of NF-kappaB activation.

Antibody to p65(RelA) for Western blots

Antibody to p65 for Western blot is included in this kit in 100 microliter aliquots (rabbit polyclonal, IgG; applicable at 1:400 dilution.  The antibody is reactive toward human, mouse, hamster, rat, canine, bovine, porcine and equine p65.  Use anti-rabbit IgG-HRP to detect the anti-p65 antibody on Western blots).  See Images and Data tab for examples of reactivity of the provided p65 antibody

Icon Image:    Structure of NF-kB p65-p50 heterodimer bound to PRDII element of ß-interferon promoter, PDB:  DOI:10.2210/pdb2i9t/pdb   NDB ID: PD0866.
  

References, p65(RelA) Translocation

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