RELA / NF-KB P65 antibody

Antigen: RELA / NF-KB P65

Clonality: Polyclonal

Host: Rabbit

Reactivity: Human, Mouse (Murine), Rat (Rattus)

Application: Enzyme Immunoassay (EIA), Electrophoretic Mobility-Shift Assay (EMSA), Immunoprecipitation (IP), Western Blotting (WB)

Catalog no.: ABIN116521

Additional Information

Characteristics: Synonyms: NF kappa B p65, NFkB p65, Transcription factor p65, Rel A, NFKB3, Nuclear factorNF-kappa-B p65 subunit, Nuclear factor of kappa light polypeptide gene enhancer inB-cells 3

Swiss-Prot: Q04206

Immunogen: Human NFkB p65 (Rel A) peptide corresponding to a region near the C-terminus of thehuman protein conjugated to Keyhole Limpet Hemocyanin (KLH)

Format: Serum

Description: NFkB was originally identified as a factor that binds to the immunoglobulin kappa lightchain enhancer in B cells. It was subsequently found in non-B cells in an inactivecytoplasmic form consisting of NFkB bound to IkB. NFkB was originally identified as aheterodimeric DNA binding protein complex consisting of p65 (RelA) and p50 (NFKB1)subunits. Other identified subunits include p52 (NFKB2), c-Rel, and RelB. The p65, cRel,and RelB subunits are responsible for transactivation. The p50 and p52 subunits possessDNA binding activity but limited ability to transactivate. p52 has been reported to formtranscriptionally active heterodimers with the NFkB subunit p65, similar to p50/p65heterodimers. The heterodimers of p52/p65 and p50/p65 are regulated by physicalinactivation in the cytoplasm by IkB-alpha. IkB-alpha binds to the p65 subunit, preventingnuclear localization and DNA binding. Low levels of p52 and p50 homodimers can alsoexist in cells.

Specificity: This antibody may react non-specifically with other proteins. Control Peptide (#R1009CP) will compete only with the specific reaction of antiserum withNFkB p65 (Rel A) subunit. Species: Human, Mouse and Rat. Others not tested.

Application Details

Application Notes: Immunoprecipitation, Immunoblotting, ELISA and supershift assays. Recommended Dilutions: This product was assayed by immunoblot and found to bereactive against NFkB p65 (Rel A) at a dilution of 1/2000 to 1/5000 followed by reactionwith Peroxidase conjugated Goat anti-Rabbit IgG (H&L) (Cat#R1364HRP). Anti-NFkB p65 (RelA) is suitable for the detection by immunoblot of human and mouse NFkB p65 (Rel A). This product was also tested in a gel supershift assay and found to be reactive against allp65 (Rel A) containing human, mouse or rat NFkB complexes using 0.5 to 1.0 ul per assay. Gel (Super) Shift Information: NFkB gel shift assays are assembled in 20 ul reactions containing 0.28 pmoles NFkB oligoin 10mM Tris (pH 7.6), 50 mM NaCl, 0.5 mM EDTA, 1.0 mM DTT, 10% glycerol. Someprocedures specify the addition of 0.05% NP-40. When using purified protein, 250-300 ngshould be sufficient to produce a gel shifted complex, while 10 ug HeLa nuclear extract isutilized. The gel shift reactions are then incubated at room temperature for 30 minutes. The

Concentration: 80.0 mg/ml (by Refractometry)

Storage: Aliquot contents and freeze at -20°C or below. Avoid repeated freezing and thawing. Dilute only prior to immediate use. Shelf life: six months from despatch.

Restrictions: For Research Use only

Images

Immunoblot of HeLa cell extract. All incubations except color development were performed using TBS supplemented with 0.1% Tween-20 at room temperature. The membrane was blocked in 5% dry milk for 2 h. After washing, a:1:5,000 dilution of the primary antibody was added to the membrane and incubated for 2 h. Washes with buffer were performed 4 times for 5' each. The immunoblot was incubated with secondary antibody (HRP Goat-a-Rabbit IgG [H&L]) diluted 1:2,000 for 1 h. Washes with TBS preceded color development.

Publications

Publications: Unlap, Jope: "Dexamethasone attenuates NF-kappa B DNA binding activity without inducing I kappa B levels in rat brain in vivo." in: Brain research. Molecular brain research, Vol. 45, Issue 1, pp. 83-9, 1997 (PubMed).

Jobin, Bradham, Russo et al.: "Curcumin blocks cytokine-mediated NF-kappa B activation and proinflammatory gene expression by inhibiting inhibitory factor I-kappa B kinase activity." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 163, Issue 6, pp. 3474-83, 1999 (PubMed).