Primary Antibodies  >  Transcription Factor Antibodies

NFkB p65 Antibody

Rabbit Polyclonal
JCYIA Product
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100 µg
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NFkB p65 Antibody Properties

Anti-NFKB p65 NLS specific (RABBIT) Antibody - 600-401-271
Target Species
Known Cross Reactivity
ELISA : 1:5,000 - 1:25,000
GelShift : 0.5 µL - 1.0 µL
IF Microscopy : 1:200
Western Blot : 1:2,000
Immunohistochemistry: 1:200
Other Dilution: User Optimized
Physical State
Liquid (sterile filtered)
Shipping Condition
Dry Ice
1.0 mg/mL by UV absorbance at 280 nm
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
0.01% (w/v) Sodium Azide
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NFkB p65 Antibody 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.
rabbit anti-NFKB p65 antibody, rabbit anti-p65 antibody, rabbit anti-NLS specific antibody, nuclear localization sequence, NFKB, NFkß, NF-kB, NF-kappaB, NFkappaB, Transcription factor p65, Nuclear factor NF-kappa-B p65 subunit, Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3, RELA, NFKB3
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.
Immunogen Type
Storage Condition
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.
Application Note
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. This antibody has been tested in ELISA, ICC, and IF.
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 NFkB heterodimer.  Therefore, the antibody selectively binds to the activated form of NFkB.  Anti-NFkB p65 NLS may react non-specifically with other proteins.
Disclaimer Note-General
This product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information. All products of animal origin manufactured by Rockland Immunochemicals are derived from starting materials of North American origin. Collection was performed in United States Department of Agriculture (USDA) inspected facilities and all materials have been inspected and certified to be free of disease and suitable for exportation. All properties listed are typical characteristics and are not specifications. All suggestions and data are offered in good faith but without guarantee as conditions and methods of use of our products are beyond our control. All claims must be made within 30 days following the date of delivery. The prospective user must determine the suitability of our materials before adopting them on a commercial scale. Suggested uses of our products are not recommendations to use our products in violation of any patent or as a license under any patent of Rockland Immunochemicals, Inc. If you require a commercial license to use this material and do not have one, then return this material, unopened to: Rockland Inc., P.O. BOX 5199, Limerick, Pennsylvania, USA.
General Reference
Delfino, F. and Walker, W.H., (1999) Hormonal regulation of the NF-kB signaling pathway. Molecular and Cellular Endocrinology  157, 1-9. Zhang G,  and Ghosh S. (2000) Molecular mechanisms of NF-kB activation induced by bacterial lipopolysaccharide through Toll-like receptors. J Endotoxin Res  6(6), 453-7. Unlap, M.T. and Jope, R.S. (1997). Dexamethasone attenuates NF-kB DNA binding activity without inducing IkB levels in rat brain in vivo. Molecular Brain Research 45: 83-89.
Specific Reference
Starkey JM, Haidacher SJ, LeJeune WS, et al. (2006) Diabetes-induced activation of canonical and noncanonical nuclear factor-kappaB pathways in renal cortex. Diabetes. May;55(5):1252-1259. Khoury J, Ibla JC, Neish AS, Colgan SP. Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation. J. Clin. Invest. 2007 Mar;117(3):703-711. Liu Q, Li J, Khoury J, et al. Adenosine signaling mediates SUMO-1 modification of IkappaBalpha during hypoxia and reoxygenation. J. Biol. Chem. 2009 May 15;284(20):13686-13695. Yu B, Chang J, Liu Y, Li J, Kevork K, Al-Hezaimi K, Graves DT, Park NH, Wang CY. (2014) Wnt4 signaling prevents skeletal aging and inflammation by inhibiting nuclear factor-?B. Nat Med. 2014 Sep;20(9):1009-17. doi: 10.1038/nm.3586. Epub 2014 Aug 10. Erratum in: Nat Med. 2015 Sep;21(9):1101.
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