V-Rel Reticuloendotheliosis Viral Oncogene Homolog A, Nuclear Factor of kappa Light Polypeptide Gene Enhancer in B-Cells 3, P65 (RELA) antibody

Antigen: V-Rel Reticuloendotheliosis Viral Oncogene Homolog A, Nuclear Factor of kappa Light Polypeptide Gene Enhancer in B-Cells 3, P65 (RELA)

Synonyms: p50, KBF1, p105, EBP-1, MGC54151, NFKB-p50, NF-kappaB, NFKB-p105, NF-kappa-B, DKFZp686C01211, NF-kB, NF-KB1, Xrel1, nfkb3, xrela, MGC69459, NFKB1, kbf1, ebp-1, MGC86262, nfkb-p50, nf-kappaB, nfkb-p105, nf-kappa-b, RELA, p65, NFKB3, MGC131774

Clonality: Polyclonal

Host: Rabbit

Reactivity: Human

Application: ELISA, Western Blotting (WB), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunofluorescence (IF)

Catalog no.: ABIN105291

Additional Information

Alternative name: NFkB p65

Gene ID: 5970

UniProt: Q04206

Immunogen: This antibody was purified from whole rabbit serum prepared by repeated immunizations with the NFkB p65 peptide corresponding to the NLS of the human protein conjugated to KLH using maleimide. A residue of cysteine was added to the amino terminal end to facilitate coupling.

Format: Liquid

Isotype: IgG

Description: NFkB 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 NFkB bound to IkB. NFkB 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 NFkB subunit p65, similar to p50/p65 heterodimers. Low levels of p52 and p50 homodimers can also exist in cells. The heterodimers of p52/p65 and p50/p65 are regulated by physical inactivation in the cytoplasm by IkB-a. IkB-a binds to the p65 subunit, preventing nuclear localization and DNA binding. IkB-a binding masks the NFkB nuclear localisation signal (NLS). A broad range of external stimuli lead to activation of NFkB and set off signalling cascades that ultimately converge on the IkB kinase (IKK) complex. Activated IKK specifically and directly phosphorylates IkB-a and this phophorylation event targets IkB-a for degradation. As a consequence, NFkB NLS is uncovered and nuclear translocation occurs.

Characteristics: Concentration Definition: by UV absorbance at 280 nm

Synonyms: p50, KBF1, p105, EBP-1, MGC54151, NFKB-p50, NF-kappaB, NFKB-p105, NF-kappa-B, DKFZp686C01211, NF-kB, NF-KB1, Xrel1, nfkb3, xrela, MGC69459, NFKB1, kbf1, ebp-1, MGC86262, nfkb-p50, nf-kappaB, nfkb-p105, nf-kappa-b, RELA, p65, NFKB3, MGC131774

Application Details

Application Notes: NFkB gel shift assays are assembled in 20µl reactions containing 0.28 pmoles NFkB 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 10µg HeLa nuclear extract is utilized. The gel shift reactions are then incubated at room temperature for 30 minutes. The complexes are resolved on 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 NFkB 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 NFkB binding in vitro: millimolar amounts of GTP and ATP, spermine, spermidine, barium or calcium ions, and µM amounts of Co+3(NH3)6.
ELISA Dilution: 1:5,000 - 1:25,000 Gel Shift Dilution: 0.5 µL - 1.0 µL
Immunohistochemistry Dilution: 1:200
IF Microscopy Dilution: 1:200
Western Blot Dilution: 1:2,000

Concentration: 1.0 mg/mL

Purification: Sterile filtered

Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2

Preservative: 0.01% (w/v) Sodium azide

Storage: Expiration date is one (1) year from date of opening. None. Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.

Research Area: Atherosclerosis, Alzheimer's Disease, Apoptosis/Necrosis, Transcription Factors, Innate Immunity

Restrictions: For Research Use only

Images

Immunochemical Rabbit anti-p65 NLS was used at a 1:200 dilution to detect p65 in (A) control DU145 cells and (B) TNF stimulated DU145 cells.  Although DU145 show relatively high basal levels of nuclear p65 staining, significant enhancement of nuclear staining is seen in panel B as evidence of translocation and availability of the NLS to be bound by the antibody.  Cultured cells shown above were formalin-fixed.  Tissue staining (not shown) were formalin-fixed, paraffin embedded followed by citrate retrieval.  Blocking and hybridization included 5% NGS.

Immunofluorescencechemistry. antibodies-online's Rabbit anti-p65 NLS was used at a 1:200 dilution to detect p65 in TNF stimulated DU145 cells. Image shown is at a 1:400 magnification. Tissue was fixed and prepared as for immunohistochemistry (see above).

Publications

Starkey, Haidacher, LeJeune et al.: "Diabetes-induced activation of canonical and noncanonical nuclear factor-kappaB pathways in renal cortex." in: Diabetes, Vol. 55, Issue 5, pp. 1252-9, 2006 (PubMed).

Abe, Hines, Zibari et al.: "Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo." in: Free radical biology & medicine, Vol. 46, Issue 1, pp. 1-7, 2008 (PubMed).

Kumar, Wu, Collier-Hyams et al.: "The bacterial fermentation product butyrate influences epithelial signaling via reactive oxygen species-mediated changes in cullin-1 neddylation." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 182, Issue 1, pp. 538-46, 2008 (PubMed).

Graff, Ettayebi, Hardy: "Rotavirus NSP1 inhibits NFkappaB activation by inducing proteasome-dependent degradation of beta-TrCP: a novel mechanism of IFN antagonism." in: PLoS pathogens, Vol. 5, Issue 1, pp. e1000280, 2009 (PubMed).

Lord, Savitsky, Sitcheran et al.: "Blimp-1/PRDM1 mediates transcriptional suppression of the NLR gene NLRP12/Monarch-1." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 182, Issue 5, pp. 2948-58, 2009 (PubMed).

Liu, Li, Khoury et al.: "Adenosine signaling mediates SUMO-1 modification of IkappaBalpha during hypoxia and reoxygenation." in: The Journal of biological chemistry, Vol. 284, Issue 20, pp. 13686-95, 2009 (PubMed).

Liu, Ju, Willmarth et al.: "Nuclear factor-kappaB enhances ErbB2-induced mammary tumorigenesis and neoangiogenesis in vivo." in: The American journal of pathology, Vol. 174, Issue 5, pp. 1910-20, 2009 (PubMed).

Sivaramakrishnan, Niranjali Devaraj: "Morin regulates the expression of NF-kappaB-p65, COX-2 and matrix metalloproteinases in diethylnitrosamine induced rat hepatocellular carcinoma." in: Chemico-biological interactions, Vol. 180, Issue 3, pp. 353-9, 2009 (PubMed).