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

p65(RelA) Translocation Assay Kit (Part No. p65-Trans)
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Price: $365.00
Availability: In Stock
Model: P65-Trans
Manufacturer: FIVEphoton Biochemicals

Available Options:
p65(RelA) Translocation Assay Kit
 
For 150 p65 translocation assays based on one assay per well of a 6-well culture dish.  Suitable for mouse, rat, hamster, canine, equine, bovine, porcine, and human.
  • 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

p65(RelA) Translocation Assay;  Background
Following stimuli that elicit the NF-KB pathway, the IKB inhibitory subunit of the NF-kappa B complex is phosphorylated, ubiquitinated and degraded, exposing nuclear targeting signals in the transcription factor subunits of the NF-kappa B complex.  The NF-kappa B transcription factor subunits (p65 and p50) with exposed nuclear import signals translocate into the nucleus and bind regulatory elements that modulate gene transcription.  Since nuclear import is a key event in NF-kappa B activation, fractionation of cells into cytoplasmic and nuclear components and detection of translocated p65 in the nuclear fraction provides a quantitative assay for p65 translocation.

This p65(RelA) Translocation Assay Kit provides all of the necessary reagents to fractionate cells into cytoplasmic and nuclear fractions, as well as a selective and high affinity antibody to detect p65 in downstream Western blots.
p65(RelA)Translocation Assay Kit Contents
  1. Cytoplasmic Fractionation Reagent (CER-1;  55 ml)
  2. Nuclear Fractionation Reagent (NER-1;  3.5 ml)
  3. DTT (sufficient to make a 1M solution with a volume of 64 microliters)
  4. Rabbit Polyclonal IgG Antibody to p65.  100 microliters provided:  Use at 1:400 dilution for Western blots

Data displaying p65(RelA) translocationclick "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.   


p65(RelA) Translocation Assay, Protocol Synopsis

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

  1. Fazal F, Bijli KM, Minhajuddin M, Rein T, Finkelstein JN, Rahman A. 2009.    Essential role of cofilin-1 in regulating thrombin-induced RelA/p65 nuclear translocation and intercellular adhesion molecule 1 (ICAM-1) expression in  endothelial cells. J Biol Chem 284: 21047-56.
  2. Fazal F, Minhajuddin M, Bijli KM, McGrath JL, Rahman A. 2007. Evidence for actin cytoskeleton-dependent and -independent pathways for RelA/p65 nuclear translocation in endothelial cells. J Biol Chem 282: 3940-50.
  3. Lim JW, Kim KH, Kim H. 2008. NF-kappaB p65 regulates nucleartranslocation of Ku70 via degradation of heat shock cognate protein 70 in pancreatic acinar AR42J cells. Int J Biochem Cell Biol 40: 2065-77.
  4. Loveridge CJ, MacDonald AD, Thoms HC, Dunlop MG, Stark LA. 2008. The proapoptotic effects of sulindac, sulindac sulfone and indomethacin are mediated by nucleolar translocation of the RelA(p65) subunit of NF-kappaB.  Oncogene 27: 2648-55.
  5. Mattioli I, Sebald A, Bucher C, Charles RP, Nakano H, et al. 2004. Transientand selective NF-kappa B p65 serine 536 phosphorylation induced by T cell costimulation is mediated by I kappa B kinase beta and controls the kinetics of p65 nuclear import. J Immunol 172: 6336-44.

Safety:  Irritant.  Avoid ingestion, eye and skin contact.
Storage:  Store antibody at 4oC.  Other contents at -20 degrees C.
Shipping Options:  Overnight Fedex domestic delivery recommended (Flat rate cost $45).  Fedex two-day domestic weekday delivery: (Flat rate cost: $27.00).  Additional shipping options are available, including international priority delivery to Asia and Europe ($85-120).  Please inquire with salessupport@fivephoton.com for international priority shipping costs to your location.

 

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Category Download Link
Protocol Manual click here
p65 Antibody Sheet click here
MSDS click here



 

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