Phospho-RELA (Ser276) Antibody

What is the RELA/NFκB p65 protein and why is phosphorylation at Ser276 significant?

RELA (p65) is a subunit of the NF-κB transcription factor complex that regulates various biological processes including inflammation, immunity, differentiation, cell growth, tumorigenesis, and apoptosis. NF-κB functions as a homo- or heterodimeric complex formed by Rel-like domain-containing proteins, with the RELA-NFKB1 heterodimer being the most abundant form . Phosphorylation at Ser276 is a key post-translational modification mediated by protein kinase A (PKA) or mitogen-stimulated kinase-1 (MSK-1), which affects the protein's transcriptional activity, nuclear localization, and interaction with co-activators . This specific phosphorylation site is part of a consensus recognition sequence (RRXS) for PKA, which has implications for its regulation and detection .

What are the common applications for Phospho-RELA (Ser276) antibodies in research?

Phospho-RELA (Ser276) antibodies are utilized in multiple experimental techniques with varying dilution requirements:

These applications help researchers investigate NF-κB signaling in various experimental contexts, though Western Blot applications especially require careful validation due to documented specificity issues .

How should I properly store and handle Phospho-RELA (Ser276) antibodies?

Phospho-RELA (Ser276) antibodies should be stored at -20°C for up to one year from the date of receipt . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and affect experimental reproducibility . Most commercial preparations are formulated in PBS containing preservatives such as 50% glycerol, 0.5% BSA, and 0.02% sodium azide . When working with these antibodies, proper aliquoting upon receipt is recommended to minimize freeze-thaw cycles. For daily experimental use, antibodies should be kept on ice and returned to storage promptly to maintain their integrity and specificity.

What cell types and treatment conditions are typically used to study RELA Ser276 phosphorylation?

RELA Ser276 phosphorylation has been studied across diverse cell types including human (L363, 1321N1, A549) and mouse (L929sA, Raw264.7, C2C12) cell lines . Researchers typically induce phosphorylation using stimuli that activate either:

• PKA pathway: LPS, cAMP elevating agents (isoproterenol, forskolin)

• MSK-1 pathway: TNF-α, PMA (phorbol esters)

For experimental design, time-course studies often involve stimulation periods ranging from 5-60 minutes, with many protocols using overnight starvation of cells prior to stimulation to reduce background phosphorylation . When designing experiments, it's important to include appropriate positive controls (stimulated samples) and negative controls (unstimulated or pathway-inhibited samples) to properly evaluate antibody performance and specificity.

What is the immunogen used to generate Phospho-RELA (Ser276) antibodies?

Commercial Phospho-RELA (Ser276) antibodies are typically generated using synthetic phosphopeptides corresponding to amino acid sequences surrounding the Ser276 residue of human p65. Specifically:

• The Bio-Techne antibody (NBP1-77807) uses "RelA/NFkB p65 peptide corresponding to an internal region near phospho Serine 276 of the human protein conjugated to Keyhole Limpet Hemocyanin (KLH)" .

• The Cell Signaling antibody (no. 3037) was "generated in rabbits using a synthetic, KLH-coupled phosphopeptide, corresponding to the residues surrounding Ser276 of human p65" .

• The St John's Labs antibody (STJ90347) uses "synthesized peptide derived from the human NF-kappaB p65 around the phosphorylation site of Ser276 at the amino acid range 249-298" .

Understanding the immunogen is critical for interpreting cross-reactivity patterns and potentially troubleshooting experimental issues, as the peptide sequence determines antibody specificity.

What are the known specificity issues with commercial Phospho-RELA (Ser276) antibodies in Western blot applications?

Multiple studies have identified significant specificity concerns with commercial anti-P-p65 Ser276 antibodies in Western blot applications. Key findings include:

• Four widely used commercial antibodies (Cell Signaling no. 3037, SAB no. 11011, Rockland no. 100-401-264, and a homemade antibody) predominantly detect bands at 130 kDa and 80 kDa rather than the expected 65 kDa band for phosphorylated p65 .

• siRNA-mediated silencing of p65 expression did not reduce these immunoreactive bands, strongly suggesting that these antibodies are not detecting p65 protein .

• Silencing of PKAcα significantly reduced the intensity of these bands, indicating cross-reactivity with other PKA-regulated proteins .

• The immunoreactive bands can be blocked with the phosphopeptide used for immunization, confirming that the cross-reactivity involves proteins with amino acid sequences homologous to the immunizing phosphopeptide .

These findings warrant extreme caution when interpreting Western blot data generated using these antibodies and suggest that researchers should implement complementary, antibody-independent approaches to verify p65 Ser276 phosphorylation.

How can researchers validate the specificity of Phospho-RELA (Ser276) antibodies in their experimental systems?

Given the documented specificity concerns, rigorous validation of Phospho-RELA (Ser276) antibodies is essential. A comprehensive validation approach should include:

• Phosphopeptide competition assays: Pre-incubating the antibody with the phosphopeptide used for immunization should abolish specific signals. This test confirms that detected bands represent specific interactions with sequences homologous to the immunizing phosphopeptide .

• siRNA knockdown experiments: Silencing p65 expression using siRNA should reduce or eliminate signals generated by truly p65-specific antibodies. If signals persist despite effective p65 knockdown (validated by probing with total p65 antibodies), cross-reactivity is likely occurring .

• Phosphatase treatment: Treating samples with lambda phosphatase before immunoblotting should eliminate signals from phospho-specific antibodies if they are truly detecting phosphorylated epitopes.

• Kinase inhibition or activation: Using specific inhibitors of PKA or MSK-1 (the kinases responsible for Ser276 phosphorylation) should reduce signal intensity, while pathway activators should increase it if the antibody is specific.

• Parallel detection methods: Use alternative techniques such as mass spectrometry or Phos-tag gels to confirm phosphorylation status independent of antibody-based detection.

What alternative experimental approaches can be used to study RELA Ser276 phosphorylation without relying solely on antibody detection?

To overcome antibody specificity limitations, researchers should consider these alternative approaches:

• Phospho-site mutant expression: Generate Ser276 to Alanine (S276A, phospho-deficient) or Ser276 to Aspartate/Glutamate (S276D/E, phospho-mimetic) mutations and compare functional outcomes with wild-type p65.

• Mass spectrometry: Immunoprecipitate p65 and analyze phosphorylation sites using targeted mass spectrometry, which can provide unambiguous identification of phosphorylated residues.

• Phos-tag SDS-PAGE: This technique causes a mobility shift in phosphorylated proteins without requiring phospho-specific antibodies, which can be detected using total p65 antibodies.

• In vitro kinase assays: Using purified PKA or MSK-1 with recombinant p65 substrate can directly demonstrate phosphorylation at Ser276 when combined with mass spectrometry.

• Proximity ligation assays: These can detect protein-protein interactions that are dependent on Ser276 phosphorylation, such as p65-CBP interaction.

• Chromatin immunoprecipitation (ChIP): To assess functional consequences of Ser276 phosphorylation on p65 binding to target gene promoters.

These approaches provide complementary evidence for Ser276 phosphorylation and its functional significance without exclusive reliance on potentially cross-reactive antibodies.

How does phosphorylation at Ser276 differ from other phosphorylation sites on RELA, and what are the comparative detection challenges?

RELA contains several phosphorylation sites with distinct functions and detection characteristics:

Unlike Ser276, detection of phosphorylation at Ser536 appears more reliable, as demonstrated by experiments showing that anti-P-p65 Ser536 antibody recognizes a 65 kDa band that comigrates with unphosphorylated p65 and is inhibited when p65 is silenced . The Ser276 residue is part of a PKA consensus recognition sequence (RRXS) that may be present in multiple PKA substrates, potentially explaining the high degree of cross-reactivity observed with anti-P-p65 Ser276 antibodies . Additionally, the structural context of Ser276 within the protein may affect epitope accessibility in different experimental conditions.

What methodological modifications can improve the reliability of immunohistochemistry experiments using Phospho-RELA (Ser276) antibodies?

While Western blot applications of Phospho-RELA (Ser276) antibodies show significant specificity issues, immunohistochemistry applications may be optimized with these methodological considerations:

For example, the Bio-Techne antibody (NBP1-77807) has been used at 1:500 dilution to detect p65 in human kidney tissue by IHC, with visualization achieved using a red precipitate signal and hematoxylin purple nuclear counterstain .

How should researchers interpret contradictory results between different detection methods for RELA Ser276 phosphorylation?

When facing contradictory results between detection methods, researchers should:

• Prioritize functional readouts: Assess whether observed biological effects align with expected outcomes of Ser276 phosphorylation (e.g., increased transcription of NF-κB target genes, enhanced p65-CBP interaction).

• Evaluate technical limitations: Consider that each method has inherent limitations:

  • Western blot may detect cross-reactive proteins

  • IHC results may be influenced by fixation and processing artifacts

  • ELISA may detect denatured epitopes not relevant to native protein

• Weigh antibody-independent evidence: Give greater weight to results from phospho-mutant studies, mass spectrometry, or Phos-tag gel analyses.

• Consider stimulus-specific effects: Different stimuli (TNF-α vs. forskolin) activate distinct kinases (MSK-1 vs. PKA) that may phosphorylate Ser276 with different kinetics or in different subcellular compartments.

• Examine subcellular localization: Ser276 phosphorylation may occur in specific cellular compartments, so nuclear/cytoplasmic fractionation before analysis may resolve apparently contradictory results.

• Report all data transparently: When publishing, clearly describe all experimental conditions, antibody validation steps, and potential limitations of the detection methods used.

What impact does the cross-reactivity of Phospho-RELA (Ser276) antibodies have on the interpretation of published literature?

The documented cross-reactivity of Phospho-RELA (Ser276) antibodies raises significant concerns about the reliability of published findings: