NCF4 Antibody

What is NCF4 and why is it important in immunological research?

NCF4 (Neutrophil Cytosolic Factor 4, 40kDa), also known as p40phox, is a critical component of the NADPH oxidase 2 (NOX2) complex responsible for reactive oxygen species (ROS) production in phagocytes. This protein plays a vital role in regulating intracellular ROS which affects B cell differentiation and immune function. Recent studies have demonstrated that NCF4 regulates the terminal differentiation of B cells to plasma cells through intracellular ROS signaling . When designing experiments involving NCF4, researchers should consider its dual role in both extracellular and intracellular ROS production, as these functions can be differentially affected by mutations.

What are the key applications of NCF4 antibodies in immunological research?

NCF4 antibodies can be utilized in multiple applications, including:

• Western Blotting (WB) to detect endogenous levels of total p40phox

• Immunohistochemistry (IHC) to visualize tissue distribution

• Immunoprecipitation (IP) for protein interaction studies

• Immunocytochemistry (ICC) and Immunofluorescence (IF) for subcellular localization

For comprehensive experimental design, researchers should select antibodies validated for their specific application and consider using multiple detection methods to cross-verify results, especially when studying NCF4's dynamic localization between NADPH complex and perinuclear regions.

How should researchers select the appropriate NCF4 antibody for their experiments?

Selection criteria should include:

• Target epitope: Choose between N-terminal, C-terminal, or internal region antibodies based on your experimental goals:

  • C-terminal antibodies (e.g., ABIN6263536) may detect full-length protein

  • PX domain-targeting antibodies are useful for studying membrane interactions

• Host species and clonality: Available options include:

  • Rabbit polyclonal antibodies for broad epitope recognition

  • Rabbit monoclonal antibodies for higher specificity

  • Mouse polyclonal antibodies for certain applications

• Validated applications: Verify the antibody has been tested in your application of interest with proper controls

How can researchers effectively study NCF4 mutations and their impact on ROS production?

To effectively study NCF4 mutations:

• Experimental design considerations:

  • Compare intracellular versus extracellular ROS production separately, as mutations like R58A affect them differently

  • Use point mutations in the PX domain (e.g., R58A) to specifically study phospholipid binding without affecting protein expression levels

  • Employ both chemical stimulants (PMA, fMLF) and physiological stimuli (phagocytosis) to assess different activation pathways

• Methodological approach:

  • Generate mouse models with specific NCF4 mutations (e.g., R58A mutation in the PtdIns3P binding site)

  • Use flow cytometry with ROS-sensitive dyes to quantify production

  • Combine with antibody detection methods to correlate ROS levels with NCF4 localization

  • Apply immunofluorescence to track subcellular localization changes in response to stimulation

• Data interpretation:

  • Consider differential effects on ROS production in different cell types (neutrophils vs. B cells)

  • Analyze both basal and stimulated ROS levels

What are the technical considerations when studying NCF4's role in B cell differentiation to plasma cells?

When investigating NCF4's role in B cell differentiation:

• Experimental system selection:

  • Use primary B cells from NCF4-mutant mice (e.g., 58A/58A Ncf4) compared to wild-type (R58/R58 Ncf4)

  • Consider chimeric B cell transfer experiments to confirm B cell-intrinsic effects

  • Employ in vitro stimulation with LPS or CD40L with anti-IgM to model differentiation

• Critical measurements:

  • Assess plasma cell formation using flow cytometry (CD19-CD138+ markers)

  • Quantify antibody-secreting cells with ELISPOT assays

  • Monitor CXCR3/CXCR4 expression on plasma cells, as NCF4 mutations alter their expression patterns

  • Measure antibody production of different isotypes (IgG1, IgG2b) which are differentially affected

• Confounding variables to control:

  • Account for effects on antigen presentation

  • Control for autoreactive T cell activation

  • Monitor germinal center formation

How should researchers approach the study of NCF4's role in inflammasome activation?

Recent findings highlight NCF4's involvement in inflammasome activation, requiring specific methodological considerations:

• Experimental approach:

  • Perform immunoprecipitation-mass spectrometry analysis of ASC to identify NCF4 interactions

  • Track NCF4 phosphorylation state during inflammasome activation

  • Monitor NCF4 puncta distribution from NADPH complex to perinuclear region

• Key parameters to measure:

  • ASC oligomerization and speck formation

  • Caspase-1 activation

  • IL-1β and IL-18 production

  • Correlation between ROS levels and inflammasome activation

• Experimental models:

  • Use NCF4-deficient mice in colorectal cancer models to assess inflammasome-IL-18-IFN-γ axis

  • Analyze CD8+ T and NK cell activation in NCF4-deficient contexts

  • Employ immunofluorescence techniques to visualize NCF4 relocalization during activation

How can researchers troubleshoot non-specific binding issues with NCF4 antibodies?

To minimize non-specific binding:

• Optimization strategies:

  • Titrate antibody concentrations (start with manufacturer's recommendations)

  • Adjust blocking conditions (5% BSA or 5% non-fat milk)

  • Increase washing steps and duration

  • Use validated antibodies with confirmed specificity for p40phox

• Controls to include:

  • Negative controls: Secondary antibody only; isotype control

  • Positive controls: Cell lines known to express NCF4 (e.g., neutrophils)

  • Competitive blocking: Pre-incubate antibody with immunizing peptide

  • NCF4 knockout or knockdown samples when possible

• Special considerations:

  • When studying tissues with low NCF4 expression, use signal amplification methods

  • For Western blotting, optimize transfer conditions for this 40kDa protein

What is the optimal protocol for co-immunoprecipitation studies involving NCF4?

For successful co-immunoprecipitation:

• Sample preparation:

  • Use gentle lysis buffers to preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain phosphorylation states

  • Consider crosslinking for transient interactions

• IP protocol optimization:

  • Select antibodies validated for IP applications (e.g., ABIN7268900)

  • For studying NOX2 complex interactions, co-IP both NCF4 and potential binding partners

  • When investigating inflammasome interactions, consider proximity ligation assays as complementary approach

• Detection strategies:

  • Use multiple antibodies targeting different epitopes for verification

  • Consider mass spectrometry to identify novel interaction partners

  • For studying NCF4-ASC interactions, look for co-localization in the perinuclear region

How should researchers address discrepancies in NCF4 detection between different antibodies?

When facing inconsistent results:

• Analysis of discrepancies:

  • Compare epitope locations: Different antibodies may detect different protein domains

  • Assess cross-reactivity with similar proteins (e.g., other NOX components)

  • Consider conformational changes affecting epitope accessibility

• Verification approaches:

  • Employ multiple antibodies targeting different regions of NCF4

  • Correlate with mRNA expression data

  • Use recombinant NCF4 protein as a positive control

  • Validate with genetic models (siRNA knockdown, CRISPR knockout)

• Resolution strategies:

  • For critical experiments, sequence verify the NCF4 in your experimental system

  • Consider using tagged NCF4 constructs as references

  • Document antibody lot numbers and experimental conditions

What are the best methodological approaches for studying NCF4 in colorectal cancer research?

Based on recent findings linking NCF4 to colorectal cancer:

• Experimental design:

  • Analyze NCF4 expression in paired tumor/normal tissues

  • Correlate expression levels with five-year survival data

  • Assess NCF4's effect on transit-amplifying and precancerous cells

• Technical implementation:

  • Use immunohistochemistry with validated NCF4 antibodies on tissue microarrays

  • Complement with Western blot analysis of fresh tissues

  • Apply multiplexed immunofluorescence to analyze NCF4 and inflammasome components simultaneously

• Functional assessment:

  • Monitor inflammasome-IL-18-IFN-γ axis activation

  • Analyze CD8+ T and NK cell frequency and activation

  • Track colorectal cancer progression in NCF4-deficient animal models

How can researchers effectively study NCF4 phosphorylation states?

To investigate NCF4 phosphorylation:

• Antibody selection:

  • Use phospho-specific antibodies when available

  • Verify specificity using phosphatase treatment controls

  • Consider generating custom phospho-antibodies for specific sites

• Analytical methods:

  • Employ Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Use 2D gel electrophoresis to resolve different phosphorylated species

  • Apply mass spectrometry to identify specific phosphorylation sites

• Functional correlation:

  • Monitor phosphorylation during NCF4 translocation from NADPH complex to perinuclear region

  • Correlate phosphorylation state with inflammasome activation

  • Assess impact on binding to PtdIns3P and subsequent ROS production

What emerging techniques should researchers consider when studying NCF4 in immune cell function?

Forward-looking methodologies include:

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize NCF4 in nanoscale subcellular structures

  • Live-cell imaging to track NCF4 translocation during cell activation

  • Correlative light-electron microscopy to study NCF4 at membrane interfaces

• Single-cell analysis:

  • Single-cell RNA-seq to correlate NCF4 expression with immune cell states

  • Mass cytometry (CyTOF) to simultaneously measure NCF4 with multiple immune markers

  • Single-cell western blotting for protein-level confirmation

• Genome editing applications:

  • CRISPR-Cas9 to generate precise NCF4 mutations (e.g., PX domain mutations)

  • Knock-in of fluorescent tags for real-time visualization

  • Creation of conditional NCF4 knockout models for tissue-specific studies