RELA / NF-kB p65 (NLS specific) antibody

Antigen: RELA / NF-kB p65 (NLS specific)

Clonality: Polyclonal

Host: Rabbit

Reactivity: Human

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

Catalog no.: ABIN117926

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: This antibody was purified from whole rabbit serum prepared by repeated immunizationswith the NFκB p65 peptide corresponding to the NLS of the human protein conjugated toKLH using maleimide. A residue of cysteine was added to the amino terminal end tofacilitate coupling. AA Sequence: Peptide Sequence: R-I-E-E-K-R-K-R-T-Y-E-T

Description: NFκB 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 NFκB bound to IκB. NFκB 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 NFκB subunit p65, similar to p50/p65heterodimers. Low levels of p52 and p50 homodimers can also exist in cells. Theheterodimers of p52/p65 and p50/p65 are regulated by physical inactivation in thecytoplasm by IκB-α. IκB-α binds to the p65 subunit, preventing nuclear localization andDNA binding. IκB- α binding masks the NFκB nuclear localisation signal (NLS). A broadrange of external stimuli lead to activation of NFκB and set off signalling cascades thatultimately converge on the IκB kinase (IKK) complex. Activated IKK specifically and directlyphosphorylates IκB-α and this phophorylation event targets IκB-α for degradation. As aconsequence, NFκB NLS is uncovered and nuclear translocation occurs.

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 NFκB heterodimer. Therefore, the antibody selectively binds to the activated form of NFκB. Anti-NFκB p65 NLS may react non-specifically with other proteins. Add. Information: Gel (Super) Shift Information: NFκB gel shift assays are assembled in 20µl reactionscontaining 0. 28 pmoles NFκB oligo in 10mM Tris (pH 7. 6), 50 mM NaCl, 0. 5 mM EDTA, 1. 0mM DTT, 10% glycerol. Some procedures specify the addition of 0. 05% NP-40. When usingpurified protein, 250-300 ng should be sufficient to produce a gel shifted complex, while10 µg HeLa nuclear extract is utilized. The gel shift reactions are then incubated at roomtemperature 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 thenegative control reaction only, as these dyes can increase the dissociation of the NFκBcomplexes. When using HeLa nuclear extract as the source of binding proteins, twosequence specific gel-shifted complexes are expected, consisting of p50/p50 homodimersand p50/p65 heterodimers. For cells expressing p52, p50, and p65, as many as foursequencespecific 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 beweakly detected. The following reagents have been observed to enhance NFκB binding invitro: millimolar amounts of GTP and ATP, spermine, spermidine, barium or calcium ions,and µM amounts of Co+3(NH3)6.

Application Details

Application Notes: Suitable for Immunohistochemistry, Immunoblotting, ELISA and supershift assays. Thisproduct was assayed by Immunoblot and found to be reactive against NFκB p65 NLS at adilution of 1: 2,000 followed by reaction with Peroxidase conjugated Affinity Purified

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

Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2 with 0.01%Sodium Azide as preservative.

Storage: Store vial at -20°C. For extended storage aliquot contents and freeze at -20°C or below. Dilute only prior to immediate use. Avoid cycles of freezing and thawing. Shelf life: One year from despatch.

Restrictions: For Research Use only

Images

Immunohistochemistry. 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 hybridizationincluded 5%

Immunofluorescencechemistry. 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).