Anti-Phospho-NF-kB p65/RelA-S311 Rabbit pAb for WB, ELISA - Q04206

Core Product Specifications and Parameters

Biological Background: Transcription factor p65 Function and Localization

• NF-kappa-B is a pleiotropic transcription factor present in almost all cell types, functioning as the endpoint of various signal transduction cascades. It forms homo- or heterodimers; the heterodimeric RELA-NFKB1 complex is the most abundant and acts as a transcriptional activator.
• RELA (p65) binds kappa-B sites in target gene promoters with distinct dimer-specific affinity and specificity, directly activating transcription and also indirectly modulating chromatin accessibility to other factors.
• In unstimulated cells, NF-kappa-B complexes are held inactive in the cytoplasm by I-kappa-B inhibitors. Upon stimulation, I-kappa-B is phosphorylated by IKKs and degraded, releasing RELA to translocate to the nucleus and initiate gene expression.
• RELA is subject to extensive post-translational modifications—including phosphorylation at multiple sites (e.g., S311), acetylation, methylation, and ubiquitination—that regulate its activity, stability, and protein interactions.
• Essential for cytokine gene expression in T-cells, highlighting its critical role in adaptive immune responses.
  Related references: PMID:15790681
• Identified as a key transcription factor governing the interferon response during SARS-CoV-2 infection. Related references: PMID:33440148
• Subcellular localization is predominantly cytoplasmic under resting conditions due to I-kappa-B masking of the nuclear localization signal; nuclear accumulation occurs rapidly after activation.
• Annotated with keywords including Phosphoprotein, Acetylation, Methylation, Ubl conjugation, Isopeptide bond, and S-nitrosylation, reflecting the intricate regulatory network controlling this master transcription factor.

Experimental Guidance and Technical Tips

• The immunogen, a synthetic phospho-peptide covering S311, is designed to generate antibodies specific for the phosphorylated form. Verify phospho-specificity by treating lysates with lambda phosphatase prior to WB.
• Suitable applications per supplier: WB and ELISA. For WB, use 20–30 µg of whole-cell lysate and a standard blocking buffer; validate band size around 60 kDa with appropriate positive controls (e.g., TNF-α stimulated HeLa cells).
• For ELISA, consider coating with the phospho-peptide and using its non-phosphorylated counterpart as a negative control to confirm site-specific binding.
• Cross-react with human and mouse; perform initial validation in relevant cell lines or tissues of these species. Induction of NF-kB signaling with stimuli such as TNF-α or PMA/ionomycin is recommended to enhance phosphorylation signals.
• Avoid harsh membrane stripping that may alter phosphorylation status; instead, use low-pH or mild detergent buffers if reprobing is required.

CamelBio: Your One-Stop Sourcing Bridge

CamelBio supports diagnostic manufacturers and research teams with integrated raw-material sourcing for every development stage, from assay concept to clinical application. This phospho-NF-kB p65 antibody fits into inflammation, immunology, and cancer biomarker workflows, and we can complement it with validated recombinant proteins, matched antibody pairs, and ancillary detection reagents. Our portfolio covers bulk monoclonal and polyclonal antibodies, rare target reagents, and custom phosphorylation-specific antibody generation to streamline your IVD kit assembly.