Phospho-NCF1 (Ser359) Antibody

What is NCF1/p47-phox and why study its phosphorylation at Ser359?

NCF1 (Neutrophil Cytosol Factor 1), also known as p47-phox, is a 47 kDa cytosolic subunit of neutrophil NADPH oxidase. This protein, along with NCF2 and membrane-bound cytochrome b558, is essential for activating latent NADPH oxidase, which produces superoxide anion during respiratory burst . Phosphorylation at Ser359 is particularly significant because it represents one of the key regulatory phosphorylation events that occurs during NADPH oxidase activation . Mutations in the NCF1 gene have been associated with chronic granulomatous disease, highlighting its clinical relevance .

What experimental applications are suitable for Phospho-NCF1 (Ser359) Antibody?

Phospho-NCF1 (Ser359) Antibody can be utilized in multiple applications:

What is the specificity of Phospho-NCF1 (Ser359) Antibody?

The Phospho-NCF1 (Ser359) Antibody specifically detects endogenous levels of p47-phox protein only when phosphorylated at Ser359 . This specificity is typically achieved through affinity purification using phospho-peptide columns or sequential chromatography on phospho- and non-phospho-peptide affinity columns . Validation experiments often show that the antibody does not cross-react with non-phosphorylated forms of NCF1 or with other phosphorylated proteins, as demonstrated in western blot analyses where competition with the antigen-specific peptide blocks detection .

How should researchers validate Phospho-NCF1 (Ser359) Antibody for their specific experimental system?

Validation of Phospho-NCF1 (Ser359) Antibody should follow these methodological steps:

• Positive and negative controls: Use cells/tissues known to have high (e.g., activated neutrophils) and low levels of phosphorylated p47-phox at Ser359 .

• Phosphatase treatment: Treat half of your positive sample with lambda phosphatase to remove phosphorylation. A true phospho-specific antibody will show reduced or no signal in the treated sample .

• Peptide competition assay: Pre-incubate antibody with phosphorylated peptide (sequence around Ser359) before application. This should block specific binding, as shown in product validation images .

• Phosphorylation induction: Stimulate cells with PMA or other activators of NADPH oxidase and observe increased detection, confirming the antibody's responsiveness to biological phosphorylation events .

• Knockout/knockdown validation: Use NCF1 knockout models or siRNA knockdown cells as negative controls to confirm specificity .

What are the optimal sample preparation techniques for detecting phosphorylated NCF1 at Ser359?

For effective detection of phosphorylated NCF1 at Ser359, researchers should follow these critical preparation steps:

• Immediate sample processing: Phosphorylation status can change rapidly; samples should be processed immediately after collection .

• Phosphatase inhibitors: Include comprehensive phosphatase inhibitor cocktails in all lysis and extraction buffers (containing sodium fluoride, sodium orthovanadate, β-glycerophosphate, and phenylmethylsulfonyl fluoride) .

• Sample lysis protocol:

  • For cell samples: Lyse in ice-cold RIPA or NP-40 buffer with phosphatase inhibitors

  • For tissue samples: Homogenize rapidly in buffer containing 1% SDS, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA with phosphatase inhibitors

• Storage considerations: Aliquot samples and store at -80°C; avoid repeated freeze-thaw cycles that can affect phosphorylation status .

• Loading control selection: When analyzing by western blotting, use total NCF1 antibody on parallel blots rather than typical housekeeping genes to normalize phosphorylation levels .

How do experimental conditions affect NCF1 phosphorylation at Ser359, and how can this be monitored?

The phosphorylation of NCF1 at Ser359 is dynamically regulated by various stimuli and conditions:

• Activation kinetics: Phosphorylation at Ser359 typically occurs within minutes of neutrophil activation and precedes NADPH oxidase assembly. Time-course experiments should include early timepoints (30 seconds to 5 minutes) to capture this dynamic process .

• Stimulus-specific responses: Different stimuli induce distinct phosphorylation patterns:

  • PMA induces strong, sustained phosphorylation

  • fMLP causes rapid but more transient phosphorylation

  • Opsonized particles show intermediate kinetics

• Methodological approach:

  • Use selective kinase inhibitors (e.g., PKC inhibitors) to identify the kinases responsible for Ser359 phosphorylation

  • Compare phosphorylation at multiple sites (Ser304, Ser328, Ser359, etc.) to understand the hierarchical phosphorylation sequence

  • Consider subcellular fractionation to monitor translocation of phosphorylated NCF1 to the membrane

• Quantitative analysis: Use densitometry analysis of western blots or quantitative ELISA to measure the relative degree of phosphorylation across different experimental conditions .

What are the key considerations when troubleshooting unexpected results with Phospho-NCF1 (Ser359) Antibody?

When encountering unexpected results with Phospho-NCF1 (Ser359) Antibody, consider these methodological approaches:

• Antibody validation issues:

  • Check lot-to-lot variation with positive control samples

  • Verify antibody storage conditions (-20°C, avoid freeze-thaw cycles)

  • Test antibody with recombinant phosphorylated and non-phosphorylated proteins

• Experimental design considerations:

  • Ensure activation conditions effectively induce phosphorylation (e.g., PMA concentration, incubation time)

  • Verify the phosphatase inhibitor cocktail is fresh and comprehensive

  • Check protein loading and transfer efficiency with total protein stains

• Technical optimization:

  • Adjust antibody concentration based on signal-to-noise ratio (1:500 to 1:2000 range for WB)

  • Optimize blocking conditions to reduce background (BSA vs. milk, concentration, time)

  • Consider enhanced detection systems for low abundance phospho-proteins

• Biological variability analysis:

  • Account for cell-type specific differences in NCF1 expression and phosphorylation

  • Consider disease state or treatment effects on basal phosphorylation levels

  • Verify experimental timing captures the phosphorylation window

How can Phospho-NCF1 (Ser359) Antibody be used to study NADPH oxidase activation mechanisms?

Phospho-NCF1 (Ser359) Antibody provides valuable insights into NADPH oxidase activation through several experimental designs:

• Stimulus-response relationship: Compare phosphorylation kinetics across different activators (PMA, fMLP, opsonized zymosan) to map activation pathways .

• Signaling pathway dissection: Use specific kinase inhibitors alongside phospho-specific antibodies to establish the hierarchy of signaling events:

  • PKC inhibitors (e.g., staurosporine, Gö6976)

  • PI3K inhibitors (e.g., wortmannin, LY294002)

  • MAPK pathway inhibitors (e.g., U0126, SB203580)

• Translocation analysis: Combine subcellular fractionation with phospho-specific detection to correlate Ser359 phosphorylation with membrane recruitment:

  • Cytosolic fraction: Pre-activation phosphorylated NCF1

  • Membrane fraction: Assembled NADPH oxidase complex components

• Structure-function studies: Use site-directed mutagenesis (S359A or S359D) in combination with phospho-antibody detection to determine the specific contribution of Ser359 phosphorylation to:

  • NCF1 conformational changes

  • Protein-protein interactions with p22phox or p67phox

  • Superoxide production capacity

What are the technical considerations for multiplexing Phospho-NCF1 (Ser359) Antibody with other markers?

When designing multiplex experiments involving Phospho-NCF1 (Ser359) Antibody, researchers should consider:

• Antibody compatibility:

  • Host species selection: Choose primary antibodies raised in different host species (e.g., rabbit anti-phospho-NCF1 with mouse anti-total NCF1)

  • Isotype consideration: Select secondary antibodies that specifically recognize different isotypes if using primary antibodies from the same host

• Sequential detection protocols:

  • Stripping and reprobing: Use mild stripping buffers to remove phospho-antibody before reprobing for total protein

  • Order of detection: Always detect phospho-epitopes first, as they are more sensitive to loss during handling

• Fluorescence multiplexing considerations:

  • Select fluorophores with minimal spectral overlap

  • Include single-stained controls for compensation

  • Consider tyramide signal amplification for low-abundance phospho-signals

• Controls for co-localization studies:

  • Include negative controls (non-phosphorylatable mutants)

  • Use positive controls (constitutively active kinase constructs)

  • Apply appropriate statistical analyses for quantifying co-localization

How should researchers interpret contradictory findings when studying NCF1 phosphorylation at Ser359?

When faced with contradictory findings regarding NCF1 phosphorylation at Ser359, researchers should systematically evaluate:

• Antibody-related factors:

  • Epitope recognition differences: Antibodies from different sources may recognize slightly different epitopes surrounding Ser359

  • Sensitivity variations: Detection thresholds differ between antibody preparations and detection methods

  • Cross-reactivity profiles: Some antibodies may detect other phosphorylated residues with similar surrounding sequences

• Experimental system differences:

  • Cell type variations: Primary neutrophils vs. cell lines (HL-60, PLB-985) show different phosphorylation kinetics

  • Species differences: Human vs. murine systems may exhibit different regulatory mechanisms

  • Activation protocols: Concentration, timing, and type of stimulus significantly affect phosphorylation patterns

• Analysis approach:

  • Data normalization methods: Normalize to total NCF1 vs. loading controls

  • Quantification techniques: Densitometry vs. flow cytometry vs. ELISA

  • Statistical treatments: Parametric vs. non-parametric testing for small sample sizes

• Integration strategy:

  • Employ multiple detection methods (WB, IF, flow cytometry)

  • Validate with functional readouts (ROS production assays)

  • Consider temporal dynamics (early vs. late phosphorylation events)

How can Phospho-NCF1 (Ser359) Antibody be used to study chronic granulomatous disease (CGD) and other NADPH oxidase-related disorders?

Phospho-NCF1 (Ser359) Antibody offers several approaches for studying CGD and related disorders:

What controls should be included when studying NCF1 phosphorylation in inflammatory disease models?

When investigating NCF1 phosphorylation in inflammatory conditions, include these essential controls:

• Technical controls:

  • Phosphatase-treated samples as negative controls

  • PMA-stimulated neutrophils as positive controls

  • Isotype control antibodies to assess non-specific binding

• Biological reference controls:

  • Age and sex-matched healthy controls

  • Disease controls (related inflammatory condition with different mechanism)

  • Time-course controls (resolution phase samples)

• Genetic controls:

  • NCF1-deficient models (when available)

  • Cells expressing phospho-mutants (S359A)

  • Kinase-deficient models relevant to NCF1 phosphorylation

• Treatment validation controls:

  • Kinase inhibitor panels (dose-response)

  • Anti-inflammatory treatments (corticosteroids, biologics)

  • ROS scavengers to link phosphorylation to functional outcomes

• Analytical controls:

  • Total NCF1 measurement in parallel samples

  • Multiple phosphorylation sites (Ser304, Ser328, Ser359, etc.)

  • Downstream signaling markers (p38 MAPK, ERK phosphorylation)