Thermo Fisher Scientific Phospho-NFkB p65 (Ser529) Monoclonal Antibody (B33B4WP), eFluor 660, eBioscience

Applications and Tested Dilution

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Product Specifications

Additional Formats:

• eFluor 450
• PE
• PE-Cyanine7
• PerCP-eFluor 710
• Request custom conjugation

Product Specific Information

The B33B4WP monoclonal antibody recognizes human NFκB p65 subunit when serine 529 is phosphorylated.
NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a transcription factor regulating genes involved in proliferation, apoptosis, immunity, and cancer. Functional NFκB is a homo- or heterodimer composed of p65 (RelA), c-Rel, RelB, p50, and p52. Its activity is regulated by IκB inhibitors that sequester NFκB in the cytoplasm.

Activation by TNF-α, IL-1β, LPS, or PMA induces IκB degradation, allowing NFκB nuclear translocation and gene regulation. Phosphorylation of p65 at Ser529 or Ser536 positively regulates NFκB activity.
The B33B4WP antibody detects a 65 kDa band in TNF-α–stimulated HeLa cell lysates.

Applications Reported: Intracellular staining followed by flow cytometric analysis.
Applications Tested: Pre-titrated for intracellular staining and flow cytometry of stimulated human peripheral blood cells. Use 5 µL (0.06 µg) per test (100 µL final volume, 10⁵–10⁸ cells/test).

Recommended Protocols:

• Protocol A: Two-step intracellular (cytoplasmic) proteins
• Protocol C: Two-step fixation/methanol
• Protocol B (nuclear proteins) not recommended

Detailed procedures available in BestProtocols® – Staining Intracellular Antigens for Flow Cytometry.

Fluorochrome Information:
eFluor® 660 is an alternative to Alexa Fluor® 647, excited at 633–647 nm and emitting at 668 nm (Red Laser).
Filtration: 0.2 µm post-manufacturing filtered.

Target Information

NF-κB p65 (RELA gene) is located on human chromosome 11. It forms heterodimers with p50 or p52 and plays a key role in transcriptional regulation of immune and inflammatory responses. Activation involves TNF-α–induced phosphorylation of IKK, leading to IκB degradation and NFκB nuclear translocation. NFκB p65 is essential for immunoglobulin κ gene transcription in mature B cells.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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