Product Pathways - NF-kappaB Signaling

Phospho-NF-KappaB p65 (Ser536) (93H1) Rabbit mAb (Alexa Fluor®488 Conjugate) #4886

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Applications	Reactivity	Sensitivity	MW (kDa)	Source	Isotype
F	H (M) (R)	Endogenous	65	Rabbit	IgG

Applications Key:  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat
Species enclosed in parentheses are predicted to react based on 100% sequence homology. Species cross-reactivity is determined by Western blot.

Specificity / Sensitivity

Phospho-NF-KappaB p65 (Ser536) (93H1) Rabbit mAb (Alexa Fluor®488 Conjugate) detects NF-kappaB p65 only when phosphorylated at serine 536. It does not cross-react with the p50 subunit or other related proteins.

Source / Purification

Monoclonal antibodies are produced by immunizing rabbits with a synthetic phospho-peptide (KLH-coupled) corresponding to residues surrounding Ser536 of human NF-kappaB p65. The antibody was conjugated to Alexa Fluor® 488 under optimal conditions with an F/P ratio of 2-6.

Description

Cell Signaling Technology Antibody conjugated to Alexa Fluor ® 488 fluorescent dye and tested in-house for direct Flow Cytometric analysis of human cells. The unconjugated antibody, #3033, reacts with Phospho-NF-KappaB p65 (Ser536) from human, mouse, and rat. CST expects that Phospho-NF-KappaB p65 (Ser536) (93H1) Rabbit mAb (Alexa Fluor®488 Conjugate) will also recognize Phospho-NF-KappaB p65 (Ser536) in these species.

Background

Transcription factors of the nuclear factor kappaB (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 an ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKK-α (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

• Willinger, T. et al. (2005) J Immunol 175, 5895-903. This article references the use of Phospho-NF-KappaB p65 (Ser536) (93H1) Rabbit mAb (Alexa Fluor®488 Conjugate) in the following applications: Flow Cytometry

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Companion Products

• 3033 Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb
• 4340 Rabbit IgG Isotype Control (Alexa Fluor^® 488 Conjugate)
• 4887 Phospho-NF-KappaB p65 (Ser536) (93H1) Rabbit mAb (Alexa Fluor® 647 Conjugate)
• 4764 NF-κB p65 (C22B4) Rabbit mAb

Rabbit Monoclonals Produced Using Epitomics® Technology, U.S. Patent No. 5,675,063.Alexa Fluor® is a registered trademark of Molecular Probes, Inc.