Phospho-NFKB1 (Ser907) Antibody

What is NFKB1 and why is phosphorylation at Ser907 significant?

NFKB1 (Nuclear Factor Kappa B Subunit 1) is a key transcription factor involved in immune response, inflammation, and cell survival pathways. Also known by several aliases including p50, KBF1, NF-kB1, NFKB-p50, and NFkappaB, it exists initially as a p105 precursor that is processed to the p50 active form . Phosphorylation at Serine 907 occurs in the p105 precursor form and plays a crucial role in regulating protein processing and function within the NF-κB signaling cascade . This specific phosphorylation site contributes to the regulation of p105 stability and its conversion to the active p50 subunit, which subsequently affects downstream gene expression involved in inflammatory responses and cell survival mechanisms.

What applications are Phospho-NFKB1 (Ser907) antibodies suitable for?

Phospho-NFKB1 (Ser907) antibodies have been validated for multiple research applications including:

• Western Blotting (WB) at dilutions of 1:500-1:1000

• Immunohistochemistry (IHC) at dilutions of 1:50-1:100

• Enzyme-Linked Immunosorbent Assay (ELISA)

These applications enable researchers to detect and quantify the phosphorylation status of NFKB1 at Ser907 in various experimental contexts, from protein lysates to fixed tissue specimens. Particularly noteworthy is the successful application in immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue, demonstrating the antibody's utility in cancer research .

How does Phospho-NFKB1 (Ser907) antibody compare to antibodies targeting other phosphorylation sites?

The NF-κB pathway involves multiple phosphorylation events at different sites, each with distinct functional implications. Phospho-NFKB1 (Ser907) antibodies specifically target the serine 907 phosphorylation site, which differs from other commercially available antibodies such as those targeting Ser893 . When designing experiments, researchers should consider that:

• Different phosphorylation sites may be activated through distinct signaling pathways

• Multiple phosphorylation sites may work synergistically or antagonistically

• Temporal dynamics of phosphorylation can vary between sites

For comprehensive pathway analysis, researchers often employ phospho-antibody arrays that can simultaneously detect multiple phosphorylation sites, including Ser872 on the related protein NFkB-p100 .

What validation steps should I take to ensure antibody specificity?

Antibody validation is critical for generating reliable data, especially given the documented issues with specificity in NF-κB research . A comprehensive validation approach should include:

• Western blot analysis confirming a single band at the appropriate molecular weight (approximately 105 kDa for the p105 precursor)

• Absence of signal in negative controls (e.g., using genetic knockout tissues/cells where available)

• Peptide competition assays to confirm binding specificity

• Cross-validation using alternative detection methods or antibodies from different sources

Research has demonstrated that even antibodies that pass initial validation tests may still produce nonspecific results, particularly in immunohistochemistry applications . The most stringent validation involves confirming absence of signal in tissues from knockout animals, though this may not always be feasible for human-specific antibodies .

What is the optimal protocol for Western blotting with Phospho-NFKB1 (Ser907) antibody?

The following protocol outlines best practices for Western blotting:

The antibody is typically supplied at 1.0mg/mL in phosphate buffered saline (without Mg2+ and Ca2+), pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol .

How should I optimize immunohistochemistry procedures using this antibody?

For optimal IHC results with paraffin-embedded tissue sections:

• Antigen Retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is generally effective, though optimization may be required for specific tissues

• Blocking: Use 10% normal serum from the species of the secondary antibody to reduce background

• Primary Antibody Application: Apply at 1:50-1:100 dilution as recommended and incubate overnight at 4°C

• Detection System: Use polymer-based detection systems for enhanced sensitivity while minimizing background

• Controls: Include positive controls (tissues known to express phosphorylated NFKB1) and negative controls (primary antibody omission)

Successful staining has been documented in human breast carcinoma tissues, providing a useful positive control reference .

How can I distinguish between specific and non-specific signals?

Distinguishing specific from non-specific signals is particularly challenging with phospho-specific antibodies. Consider the following approaches:

• Molecular Weight Verification: For Western blots, ensure the detected band corresponds to the expected molecular weight (105 kDa for p105)

• Dephosphorylation Controls: Treat duplicate samples with lambda phosphatase prior to analysis; the phospho-specific signal should disappear

• Stimulation/Inhibition Controls: Include samples treated with known pathway activators or inhibitors to demonstrate biological responsiveness

• Comparison with Total Protein: Run parallel blots with antibodies against total (non-phospho-specific) NFKB1 to verify protein presence

• Multiple Detection Methods: Cross-validate results using different techniques (e.g., WB, IHC, ELISA)

Research has shown that antibodies raised against the nuclear localization signal regions of NF-κB proteins often exhibit non-specific binding, so particular caution is warranted when the epitope includes or is near these regions .

What are common pitfalls when using phospho-specific NFKB1 antibodies?

Several challenges specific to phospho-NFKB1 antibodies warrant attention:

• Rapid Dephosphorylation: Phosphorylation states can rapidly change during sample processing; immediate denaturation and inclusion of phosphatase inhibitors are essential

• Context-Dependent Phosphorylation: Phosphorylation status may vary significantly between cell types and stimulation conditions

• Epitope Masking: Protein-protein interactions may conceal the phosphorylated epitope, reducing detection efficiency

• Antibody Cross-Reactivity: Many antibodies exhibit cross-reactivity with structurally similar phosphorylation sites on related proteins

• Inconsistent Validation Standards: Studies have revealed that many antibodies purported to be specific fail rigorous validation tests using genetic knockout models

Many claims regarding NF-κB activity, particularly in neuronal studies, have been based on antibodies that do not pass stringent specificity tests, underscoring the importance of careful validation .

How should I interpret contradictory results between Western blot and immunohistochemistry?

Discrepancies between Western blot and IHC results are not uncommon and may arise from:

• Epitope Accessibility: The three-dimensional protein structure in fixed tissues might conceal the phosphorylated epitope

• Tissue Processing Effects: Formalin fixation can affect phospho-epitopes differently than sample preparation for Western blots

• Cellular Heterogeneity: IHC may reveal phosphorylation in specific cell subpopulations that become diluted in whole-tissue Western blot samples

• Antibody Performance Variation: Some antibodies perform better in denatured conditions (Western blot) than in partially native states (IHC)

• Background Issues: IHC may exhibit higher background staining that complicates interpretation

When encountering contradictory results, consider complementary approaches such as immunofluorescence, phospho-flow cytometry, or mass spectrometry to resolve the discrepancy.

How does Ser907 phosphorylation relate to other NFKB1 regulatory mechanisms?

Serine 907 phosphorylation represents one of multiple regulatory modifications that govern NFKB1 function:

• Processing Regulation: Phosphorylation at Ser907 influences the processing of p105 to p50, affecting the availability of active transcription factor

• Pathway Cross-talk: This phosphorylation site may integrate signals from multiple upstream kinases, positioning it as a potential convergence point in cellular signaling

• Temporal Dynamics: The phosphorylation status at Ser907 likely exhibits temporal patterns that differ from other sites such as Ser893 , potentially allowing for nuanced regulation

• Subcellular Localization Effects: Phosphorylation may influence protein-protein interactions and subcellular trafficking of NF-κB components

Research into site-specific phosphorylation events continues to reveal increasingly complex regulatory mechanisms that fine-tune NF-κB signaling in different cellular contexts.

What methodological approaches can enhance detection sensitivity and specificity?

Advanced methodological approaches to improve phospho-NFKB1 detection include:

• Proximity Ligation Assay (PLA): Allows visualization of protein interactions and modifications with single-molecule sensitivity in situ

• Phospho-proteomic Mass Spectrometry: Provides unbiased, comprehensive analysis of phosphorylation sites without antibody limitations

• CRISPR-based Validation: Generating epitope-modified cell lines using CRISPR/Cas9 for definitive antibody validation

• Multiplexed Detection Systems: Technologies such as NFkB Phospho Antibody Arrays allow simultaneous detection of multiple phosphorylation sites

• Quantitative Image Analysis: Advanced image analysis algorithms can improve quantification of immunohistochemical or immunofluorescence staining

These approaches can be particularly valuable when traditional methods yield ambiguous results or when studying complex signaling dynamics.

What are the implications of NFKB1 Ser907 phosphorylation in disease models?

Emerging research suggests significant implications of NFKB1 phosphorylation in various disease contexts:

• Cancer Biology: Immunohistochemical studies have demonstrated the presence of phosphorylated NFKB1 (Ser907) in breast carcinoma tissues, suggesting potential involvement in cancer progression

• Inflammatory Disorders: Aberrant NF-κB signaling contributes to various inflammatory conditions, with phosphorylation serving as a potential biomarker and therapeutic target

• Neurodegenerative Diseases: Although many studies of NF-κB in neuronal contexts require re-evaluation due to antibody specificity concerns , site-specific phosphorylation may still play important roles in neuroinflammation

• Therapeutic Development: Understanding the functional consequences of specific phosphorylation events may guide the development of targeted therapeutics that modulate NF-κB activity

Further research using well-validated reagents is needed to fully elucidate the role of Ser907 phosphorylation in these disease contexts.

What are the optimal storage conditions for maintaining antibody performance?

For maximum stability and performance retention:

• Long-term Storage: Store at -20°C or -80°C as recommended by manufacturers

• Working Aliquots: Prepare small working aliquots to avoid repeated freeze-thaw cycles

• Thawing Procedure: Thaw aliquots at 4°C rather than room temperature

• Buffer Composition: The antibody is typically supplied in phosphate buffered saline with 50% glycerol and 0.02% sodium azide to maintain stability

• Contamination Prevention: Use sterile technique when handling antibody solutions to prevent microbial contamination

Avoid repeated freeze-thaw cycles as these can significantly reduce antibody performance and lead to increased background signal .

How can I optimize experiment planning to maximize antibody utility?

Strategic experimental planning can maximize the utility of valuable antibody reagents:

Careful planning not only preserves valuable reagents but also enhances data quality and reproducibility.