V-Rel Reticuloendotheliosis Viral Oncogene Homolog A (Avian) (RELA) antibody

Antigen: V-Rel Reticuloendotheliosis Viral Oncogene Homolog A (Avian) (RELA)

Synonyms: p50, KBF1, p105, EBP-1, MGC54151, NFKB-p50, NF-kappaB, NFKB-p105, NF-kappa-B, DKFZp686C01211, NF-kB, NF-KB1, NFKB1, kbf1, ebp-1, MGC86262, nfkb-p50, nf-kappaB, nfkb-p105, nf-kappa-b

Clonality: Polyclonal

Host: Rabbit

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

Application: ELISA, Western Blotting (WB), Immunoprecipitation (IP)

Catalog no.: ABIN233796

Additional Information

Alternative name: NFKB p65 (Rel A)

Immunogen: NF kappaB p65 (Rel A) peptide corresponding to a region near the C-terminus of the human protein conjugated to Keyhole Limpet Hemocyanin (KLH).

Format: Liquid (sterile filtered)

Description: NF kappaB was originally identified as a factor that binds to the immunoglobulin kappa light chain enhancer in B cells. It was subsequently found in non-B cells in an inactive cytoplasmic form consisting of NF kappaB bound to IkappaB. NFkappaB was originally identified as a heterodimeric 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 possess DNA binding activity but limited ability to transactivate. p52 has been reported to form transcriptionally active heterodimers with the NF kappaB subunit p65, similar to p50/p65 heterodimers. The heterodimers of p52/p65 and p50/p65 are regulated by physical inactivation in the cytoplasm by I kappaB-alpha. IkappaB-alpha binds to the p65 subunit, preventing nuclear localization and DNA binding. Low levels of p52 and p50 homodimers can also exist in cells. Gel (Super) Shift Information: NF kappaB gel shift assays are assembled in 20ul reactions containing 0.28 pmoles NFkappaB oligo in 10mM Tris (pH 7.6), 50 mM NaCl, 0.5 mM EDTA, 1.0 mM DTT, 10% glycerol. Some procedures specify the addition of 0.05% NP-40. When using purified protein, 250-300 ng should be sufficient to produce a gel shifted complex, while 10ug HeLa nuclear extract is utilized. The gel shift reactions are then incubated at room temperature for 30 minutes. The complexes are resolved on a Tris-Glycine acrylamide gels. Loading dye containing bromophenol blue and xylene cyanol should be added to the negative control reaction only, as these dyes can increase the dissociation of the NF kappaB complexes. When using HeLa nuclear extract as the source of binding proteins, two sequence-specific gel-shifted complexes are expected, consisting of p50/p50 homodimers and p50/p65 heterodimers. For cells expressing p52, p50, and p65, as many as four sequence-specific gel-shifted complexes could be observed (p52/p52, p50/p50, p52/p65, p50/p65), and if high levels of p65 are present, the p65/p65 homodimer may also be weakly detected. The following reagents have been observed to enhance NF kappaB binding in vitro: millimolar amounts of GTP and ATP, spermine, spermidine, barium or calcium ions, and uM amounts of Co +3 (NH 3 ) 6 .

Application Details

Application Notes: Recommended Dilutions: This product was assayed by immunoblot and found to be reactive against NF kappaB p65 (Rel A) at a dilution of 1:2000 to 1:5000 followed by reaction with Peroxidase conjugated Affinity Purified anti-Rabbit IgG [H&L] (Goat) code ABIN101990. Anti-NF kappaB p65 (Rel A) is suitable for the detection by immunoblot of human and mouse NF kappaB p65 (Rel A). This product was also tested in a gel supershift assay and found to be reactive against all p65 (Rel A) containing human and mouse NF kappaB complexes using 0.5 to 1.0 ul per assay. Optimal titers for other applications should be determined by the researcher. Suitable for immunoprecipitation, immunoblotting, ELISA and supershift assays.

Concentration: 90.0 mg/ml (by Refractometry)

Buffer: Stabilizer: None. Preservative: 0.01% (w/v) Sodium Azide. Buffer: None.

Storage: Store vial at -20° C or below prior to opening. Dilute only prior to immediate use. For extended storage, aliquot contents and freeze at -20° C or below. Avoid cycles of freezing and thawing. Expiration date is six (6) months from date of opening product. Figure. Immunoblot of HeLa cell extract. A predominant band ~65 kDa (arrowhead) corresponding to NFb p65 Rel A is observed. 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.

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 tempera-ture. 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: Miller, Cheshire, Raab-Traub: "Interaction of tumor necrosis factor receptor-associated factor signaling proteins with the latent membrane protein 1 PXQXT motif is essential for induction of epidermal growth factor receptor expression." in: Molecular and cellular biology, Vol. 18, Issue 5, pp. 2835-44, 1998 (PubMed).

Feng, Cheng, Hsia et al.: "NF-kappaB inducible genes BCL-X and cyclin E promote immature B-cell proliferation and survival." in: Cellular immunology, Vol. 232, Issue 1-2, pp. 9-20, 2005 (PubMed).

Lou, Kaplowitz: "Glutathione depletion down-regulates tumor necrosis factor alpha-induced NF-kappaB activity via IkappaB kinase-dependent and -independent mechanisms." in: The Journal of biological chemistry, Vol. 282, Issue 40, pp. 29470-81, 2007 (PubMed).

Begley, Kasina, Mehra et al.: "CXCL5 promotes prostate cancer progression." in: Neoplasia (New York, N.Y.), Vol. 10, Issue 3, pp. 244-54, 2008 (PubMed).

Yoon, Korade, Carter: "Protein kinase A-induced phosphorylation of the p65 subunit of nuclear factor-kappaB promotes Schwann cell differentiation into a myelinating phenotype." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 28, Issue 14, pp. 3738-46, 2008 (PubMed).