New England Biolabs Canada
 
XP Monoclonal Antibody

Product Pathways - NF-kB Signaling

NF-κB p65 (D14E12) XP® Rabbit mAb #8242

Item# Description List Price Web Price Qty
8242S NF-KappaB p65 (D14E12) XP® Rabbit mAb - 100 µl $432.00
$388.80
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8242T NF-κB p65 (D14E12) XP® Rabbit mAb - 20 µl $180.00
$162.00
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
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VIEW COMPANION PRODUCTS HIDE COMPANION PRODUCTS
Application Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W Human, Mouse, Rat, Hamster, Monkey, Dog Endogenous 65 Rabbit IgG
IP
IHC-P
IF-IC
F
ChIP
ChIP-seq

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, IHC-P=Immunohistochemistry (Paraffin), IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, ChIP=Chromatin IP, ChIP-seq=Chromatin IP-seq

Directions For Use

For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP.

This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

Specificity / Sensitivity

NF-κB p65 (D14E12) XP® Rabbit mAb recognizes endogenous levels of total NF-κB p65/RelA protein. It does not cross react with other NF-κB/Rel family members.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu498 of human NF-κB p65/RelA protein.

Western Blotting

Western Blotting

Western blot analysis of HeLa cell extracts, untreated (-) or NF-κB p65 knock-out (+), using NF-κB p65 (D14E12) XP® Rabbit mAb, #8242 (upper) or β-actin (13E5) Rabbit mAb #4970 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis using NF-κB p65 (D14E12) XP® Rabbit mAb on SignalSlide® NF-κB p65 IHC Controls #12873 (paraffin-embedded HCT116 cells, untreated (left) or treated with hTNF-α #8902 (right)).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human chronic cholecystitis using NF-κB p65 (D14E12) XP® Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HT-1080 cells, untreated (left) or treated with hTNF-α #8902 (20 ng/ml, 20 min) (right), using NF-κB p65 (D14E12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HeLa cells using NF-κB p65 (D14E12) XP® Rabbit mAb (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).


Chromatin IP-seq

Chromatin IP-seq

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IL-8, a known target gene of NFκB (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by Real-Time PCR using SimpleChIP® Human IκBα Promoter Primers #5552, human IL-8 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Background

Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).

  1. Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.
  2. Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.
  3. Haskill, S. et al. (1991) Cell 65, 1281-9.
  4. Thompson, J.E. et al. (1995) Cell 80, 573-82.
  5. Whiteside, S.T. et al. (1997) EMBO J 16, 1413-26.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Scherer, D.C. et al. (1995) Proc Natl Acad Sci USA 92, 11259-63.
  8. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  9. Senftleben, U. et al. (2001) Science 293, 1495-9.
  10. Coope, H.J. et al. (2002) EMBO J 21, 5375-85.
  11. Xiao, G. et al. (2001) Mol Cell 7, 401-9.
  12. Kozako, T. et al. (2015) Sci Rep 5, 11345.

Application References

  • Niederreiter, L. et al. (2013) J Exp Med 210, 2041-56. Applications: Western Blotting.
  • Jacamo, R. et al. (2014) Blood 123, 2691-702. Applications: IF-IC (In Cells), Western Blotting.

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!


 

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