p65 (NFkB) antibody

Antigen: p65 (NFkB)

Clonality: Polyclonal

Host: Rabbit

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

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

Catalog no.: ABIN105291

Additional Information

Immunogen: This antibody was purified from whole rabbit serum prepared by repeated immunizations with the NF kappaB 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 (sterile filtered)

Specificity: This affinity purified antibody is directed against the nuclear localization sequence (NLS) NLS of human p65 and is useful in determining its presence in various assays. The epitope recognized overlaps the NLS of the p65 subunit of the NF kappaB heterodimer. Therefore, the antibody selectively binds to the activated form of NFkappaB. Anti-NFkappaB p65 NLS may react non-specifically with other proteins.

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 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 NFkB complexes. When using HeLa nuclear extract as the source of binding proteins, two sequencespecific 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 sequencespecific 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. Recommended Dilutions: ELISA:1:5,000 - 1:25,000 WB:1:2,000 IHC:1:200 Gel Shift: 0,5µl-1,0 µl IF:1:200 OTHER: User Optimized

Concentration: 1.0 mg/ml (by UV absorbance at 280 nm)

Purification: 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. 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 I kappaB-alpha. IkappaB-alpha binds to the p65 subunit, preventing nuclear localization and DNA binding. IkappaB-alpha binding masks the NFkappaB nuclear localisation signal (NLS). A broad range of external stimuli lead to activation of NFkappaB and set off signalling cascades that ultimately converge on the I kappaB kinase (IKK) complex. Activated IKK specifically and directly phosphorylates I kappaB-alpha and this phophorylation event targets IkappaB-alpha for degradation. As a consequence, NFkappaB NLS is uncovered and nuclear translocation occurs.

Buffer: Stabilizer: None. Preservative: 0.01% (w/v) Sodium Azide. Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2.

Storage: Store vial at -20° C or below prior to opening. Dilute only prior to immediate use. 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.

Research Area: Cancer, Apoptosis/Necrosis, Alzheimer's Disease, Signaling

Restrictions: For Research Use only

Images

Immunohistochemistry. antibodies-online's 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. Tissue was 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

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).