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.
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 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.
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