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Product Pathways - NF-kB Signaling

NF-κB p65 (L8F6) Mouse mAb #6956

Item# Description List Price Web Price Qty
6956S NF-KappaB p65 (L8F6) Mouse mAb - 100 µl $383.00
$344.70
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6956T NF-κB p65 (L8F6) Mouse mAb - 20 µl $163.00
$146.70
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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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8112S SignalStain® Antibody Diluent - 25 ml $59.00
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8125P SignalStain® Boost IHC Detection Reagent (HRP, Mouse) - 1 ml $57.00
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VIEW COMPANION PRODUCTS HIDE COMPANION PRODUCTS
Application Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W Human, Mouse, Rat, Hamster, Monkey, Mink, Bovine, Dog, Pig Endogenous 65 Mouse IgG2b
IP
IHC-P
IF-IC
F
ChIP

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

Directions For Use

For optimal ChIP results, use 10 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 (L8F6) Mouse mAb recognizes endogenous levels of total NF-κB p65 protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human NF-κB protein.

Western Blotting

Western Blotting

Western blot analysis of HeLa cell extracts, untreated (-) or NF-κB p65 knock-out (+), using NF-κB p65 (L8F6) Mouse mAb #6956 (upper) or β-actin (13E5) Rabbit mAb #4970 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® NF-κB p65 siRNA I #6261 (+), using NF-κB p65 (L8F6) Mouse mAb (upper) or α-Tubulin (11Η10) Rabbit mAb #2125 (lower). The NF-κB p65 (L8F6) Mouse mAb confirms silencing of NF-κB p65 expression, while the α-Tubulin (11Η10) Rabbit mAb is used as a loading control.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using NF-κB p65 (L8F6) Mouse mAb.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of human chronic cholecystitis tissue using NF-κB p65 (L8F6) Mouse mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded OVCAR8 cell pellets treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (left) or treated with SignalSilence® NF-κB p65 siRNA I #6261 (right), using NF-κB p65 (L8F6) Mouse mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (right), using NF-κB p65 (L8F6) Mouse mAb.


IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 20 min; right), using NF-κB p65 (L8F6) Mouse 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 (L8F6) Mouse mAb (blue) compared to a nonspecific negative control antibody (red).

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (L8F6) Mouse 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.

Application References

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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