Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-NF-κB p65 (Ser536) Sandwich ELISA Antibody Pair #7834

Kit Includes Volume Cap Color
P-NF-kB p65 (s536) Capture Ab (100X) 0.4 ml Pink
NF-kB p65 Detection Ab (100X) 0.4 ml Blue
Anti-rabbit IgG, HRP-linked Antibody 0.04 ml Red

Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Description

CST's PathScan® Phospho-NF-κB p65 (Ser536) Sandwich ELISA Antibody Pair is being offered as an economical alternative to our PathScan®Phospho-NF-κB p65 (Ser536) Sandwich ELISA Kit #7173. Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Phospho-NF-κB p65 Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by a NF-κB p65 (Ser536) Detection Antibody and anti-Rabbit IgG, HRP conjugated antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Phospho-NF-κB p65 (Ser536) protein.*Antibodies in this kit are custom formulations specific to the kit.

Specificity / Sensitivity

For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Sandwich ELISA

Sandwich ELISA

The relationship between lysate protein concentration from untreated and TNF-α and IL-1β treated HeLa cells and the absorbance at 450 nm using PathScan® Phospho-NF-κB p65 (Ser536) Sandwich ELISA Antibody Pair #7834 is shown. HeLa cells were treated with TNF-α and IL-1β for 5 minutes at 37ºC and then lysed.

Background

Transcription factors of the nuclear factor κ B (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 is then translocated 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

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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