NCF2 Antibody, FITC conjugated

What is NCF2 and why is it an important research target?

NCF2 (Neutrophil Cytosolic Factor 2), also known as p67-phox, is a cytosolic component of the NADPH oxidase complex essential for reactive oxygen species (ROS) production in phagocytes. It plays a critical role in innate immunity and has been implicated in various immune-related pathologies, particularly systemic lupus erythematosus (SLE). Research has established that the NCF2 gene predisposes to lupus, with point mutations potentially causing reduced NADPH oxidase activity . This connection between oxidative stress and autoimmunity makes NCF2 a valuable research target for understanding immune dysregulation mechanisms.

What are the fundamental characteristics of FITC-conjugated antibodies?

FITC (Fluorescein Isothiocyanate) is an amine-reactive fluorochrome that covalently binds to amino groups of antibodies. The resulting conjugates emit green fluorescence (peak emission ~520 nm) when excited with blue light (~495 nm). The conjugation process involves dialyzing purified antibody against FITC labeling buffer to remove free NH4+ ions and raise pH to 9.2, followed by reaction with FITC dissolved in anhydrous DMSO . FITC-conjugated antibodies typically require storage at -20°C or -80°C in buffer systems containing glycerol and appropriate preservatives to maintain stability and prevent repeated freeze-thaw cycles .

How does NCF2 function within the NADPH oxidase complex?

NCF2 functions as an activator protein within the NADPH oxidase complex, which is responsible for the respiratory burst in phagocytes. When activated, cytosolic components including NCF2 (p67-phox) translocate to the membrane and associate with membrane-bound components to form the active enzyme complex. This assembled complex catalyzes the production of superoxide anions, which are critical for pathogen killing. Studies using NCF2-null mice demonstrate that complete absence of NCF2 leads to elimination of NADPH oxidase activity, resulting in susceptibility to infections like Aspergillus fumigatus pneumonia, characteristic of chronic granulomatous disease . Understanding this mechanism is crucial for interpreting experimental results when using NCF2 antibodies in research.

What are the validated applications for NCF2 antibody, FITC conjugated?

Based on available research data, FITC-conjugated NCF2 antibodies have been validated for several applications:

When designing experiments, researchers should verify the specific validation data for their particular antibody lot, as reactivity can vary between manufacturers and even between lots from the same manufacturer .

How can NCF2 antibody, FITC conjugated be used to study lupus pathogenesis?

NCF2 antibodies are valuable tools for investigating the role of NADPH oxidase in lupus pathogenesis. Research has shown that even haploinsufficiency of NCF2 can accelerate lupus development in susceptible animal models. To study this connection, researchers can employ FITC-conjugated NCF2 antibodies in flow cytometry to analyze NCF2 expression levels in different immune cell populations from lupus patients or animal models.

Experimental approaches should include:

• Comparing NCF2 expression levels between healthy controls and lupus patients

• Correlating NCF2 expression with disease activity indices

• Examining the impact of NCF2 deficiency on neutrophil extracellular trap (NET) formation

• Analyzing the relationship between NCF2 expression and type I interferon responsive gene expression profiles

Studies have demonstrated that NCF2-null and even NCF2-haploinsufficient mice on lupus-prone backgrounds (e.g., NZM 2328) develop accelerated lupus with significant kidney disease, characterized by hyperactive B and T cell compartments and increased type I interferon gene expression .

What controls should be included when using FITC-conjugated NCF2 antibodies?

Proper experimental controls are essential for generating reliable data with FITC-conjugated NCF2 antibodies:

• Isotype controls: Include FITC-conjugated IgG (matching the host species and isotype of your NCF2 antibody) to control for non-specific binding

• Negative controls:

  • Unstained samples

  • Samples stained with irrelevant FITC-conjugated antibodies

  • Samples from NCF2 knockout models (when available)

• Positive controls:

  • Samples known to express high levels of NCF2 (e.g., activated neutrophils)

  • Recombinant NCF2 protein (for techniques like ELISA)

• Fluorescence minus one (FMO) controls: Particularly important for multicolor flow cytometry to establish proper gating strategies

• Blocking controls: Pre-incubation with unconjugated NCF2 antibody to confirm specificity

How should FITC-conjugated NCF2 antibodies be stored to maintain optimal activity?

Optimal storage conditions are critical for maintaining antibody performance:

Research indicates that FITC-conjugated antibodies should be protected from repeated freeze-thaw cycles, as this can lead to significant loss of fluorescence intensity and binding capacity . The presence of glycerol in the storage buffer helps prevent freeze damage while maintaining protein stability.

What is the optimal fluorochrome-to-protein ratio for FITC-conjugated NCF2 antibodies?

The fluorochrome-to-protein (F:P) ratio is critical for optimal performance of FITC-conjugated antibodies. For NCF2 antibodies:

• Optimal F:P ratio: Typically between 3:1 and 8:1 for FITC conjugates

• Too low F:P ratio: Results in insufficient fluorescence signal

• Too high F:P ratio: Can cause fluorescence quenching, increased non-specific binding, and altered antibody binding affinity

After conjugation, the F:P ratio should be determined by measuring the absorbance of the conjugate at 280 nm (protein) and 495 nm (FITC). The protocol for FITC conjugation to antibodies involves careful control of reaction conditions, including pH (optimally 9.2), temperature, and FITC concentration, to achieve appropriate labeling .

Researchers should be aware that commercially available NCF2-FITC antibodies undergo quality control to ensure appropriate F:P ratios, with protein G purification typically achieving >95% purity, contributing to consistent performance across experiments .

How can photobleaching of FITC-conjugated NCF2 antibodies be minimized?

FITC is particularly susceptible to photobleaching, which can significantly impact experimental results. To minimize this effect:

• Sample preparation:

  • Add anti-fade reagents to mounting media for microscopy applications

  • Prepare samples immediately before analysis

  • Use fresh antibody preparations for critical experiments

• During experiments:

  • Minimize exposure to excitation light

  • Use neutral density filters to reduce excitation intensity

  • For microscopy, use shorter exposure times with higher camera gain when possible

  • For flow cytometry, optimize laser power to the minimum required for adequate signal

• Equipment considerations:

  • Ensure proper alignment of light sources and filters

  • Use shutters or automated systems that limit light exposure to acquisition periods

  • Consider newer generation fluorochromes with greater photostability for particularly sensitive applications

These practices are particularly important when analyzing NCF2 expression in tissue sections or performing quantitative analyses where signal intensity is directly correlated with expression levels .

How can NCF2 expression be quantitatively assessed in different immune cell populations?

Quantitative assessment of NCF2 expression across immune cell populations requires sophisticated experimental approaches:

• Multiparameter flow cytometry:

  • Combine FITC-conjugated NCF2 antibodies with lineage markers for neutrophils, monocytes, and lymphocytes

  • Use standardized beads to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF) for absolute quantification

  • Apply compensation matrices to correct for spectral overlap when using multiple fluorochromes

• Imaging cytometry:

  • Provides spatial information about NCF2 distribution within cells while maintaining quantitative capacity

  • Can differentiate between cytoplasmic and membrane-associated NCF2 during cell activation

• qPCR validation:

  • Complement protein-level data with mRNA quantification

  • Use primers specific to NCF2 (e.g., sense: 5'-GCGCTAGGCTGGGACCTTGAAGCC-3', antisense: 5'-GTCTTGAAGAAGGGCAGTGATAAC-3')

  • Normalize to appropriate housekeeping genes like β-actin

• Single-cell analysis:

  • Correlate NCF2 expression with functional outputs at the single-cell level

  • Particularly valuable for heterogeneous populations like tumor-infiltrating immune cells

Research has demonstrated that NCF2 expression varies significantly between immune cell populations and can be altered in disease states, making quantitative assessment crucial for understanding its role in pathogenesis .

How does NCF2 haploinsufficiency impact immune function and how can this be studied?

NCF2 haploinsufficiency has significant implications for immune function, particularly in the context of autoimmunity. Research strategies to investigate this include:

• Animal models:

  • Studies with NCF2-null and NCF2-haploinsufficient mice on both normal (C57BL/6) and lupus-prone (NZM 2328) backgrounds

  • Comparative analysis of immune parameters and disease progression

• Functional assays:

  • Respiratory burst assays to measure ROS production

  • NET formation visualization and quantification

  • B and T cell activation marker analysis

  • Cytokine production profiling

• Molecular approaches:

  • Analysis of type I interferon-responsive gene expression

  • Evaluation of NADPH oxidase assembly and function

  • Assessment of redox-sensitive signaling pathways

Research has demonstrated that even 50% reduction in NCF2 (haploinsufficiency) on a lupus-prone background accelerates lupus development with increased immune activation markers, highlighting a gene-dose effect . This finding has particular relevance for human patients with NCF2 variants that may not completely abolish function but reduce it significantly.

What are the latest research findings on the relationship between NCF2 function and neutrophil extracellular trap (NET) formation?

Recent research has revealed surprising findings about the relationship between NCF2/NADPH oxidase function and NET formation:

These findings highlight the complex role of NCF2 in immune regulation beyond its canonical function in ROS production and suggest that therapeutic targeting of NCF2/NADPH oxidase should consider these nuanced effects .

What strategies can address weak or inconsistent signals when using FITC-conjugated NCF2 antibodies?

When faced with weak or inconsistent signals, researchers should consider the following optimization strategies:

• Antibody concentration optimization:

  • Titrate antibody to determine optimal concentration

  • Test range from 0.1-10 μg/ml for most applications

  • Create a signal-to-noise ratio curve to identify optimal concentration

• Sample preparation refinement:

  • Ensure proper fixation and permeabilization for intracellular staining

  • Optimize blocking conditions to reduce background

  • Consider alternative epitope retrieval methods for FFPE tissue sections

• Technical adjustments:

  • Increase incubation time at 4°C (overnight if necessary)

  • Add 0.1% saponin to maintain permeabilization during incubation

  • Use signal amplification systems if needed

• Fluorochrome considerations:

  • For samples with high autofluorescence, consider alternative fluorochromes

  • Check for potential quenching due to high local concentrations

  • Verify conjugate stability and age

• Instrument settings:

  • Optimize PMT voltage or gain for FITC channel

  • Ensure proper filter sets are being used (excitation ~495 nm, emission ~520 nm)

  • Validate with positive controls known to express high levels of NCF2

How can researchers distinguish between specific and non-specific binding of FITC-conjugated NCF2 antibodies?

Distinguishing specific from non-specific binding is critical for accurate data interpretation:

• Blocking optimization:

  • Evaluate different blocking agents (BSA, normal serum, commercial blockers)

  • Titrate blocking agent concentration (typically 1-10%)

  • Include blocking steps for both Fc receptors and non-specific binding sites

• Control implementation:

  • Use isotype controls matched to the NCF2 antibody's host species and isotype

  • Include absorption controls (pre-incubate antibody with recombinant NCF2)

  • Apply fluorescence-minus-one (FMO) controls for multicolor experiments

• Washing protocol refinement:

  • Increase number of washes after antibody incubation

  • Add low concentrations of detergent (0.05% Tween-20) to wash buffers

  • Ensure complete buffer exchange during washing steps

• Cross-reactivity assessment:

  • Test antibody on samples known to lack NCF2 expression

  • Verify species reactivity (anti-human NCF2 antibodies may cross-react with proteins from other species)

  • Check for potential cross-reactivity with other NADPH oxidase components

What experimental approaches can resolve contradictory findings when studying NCF2 function in different disease models?

Resolving contradictory findings requires systematic experimental approaches:

• Model system standardization:

  • Ensure genetic background consistency in animal models

  • Control for age, sex, and environmental factors

  • Document detailed experimental conditions for reproducibility

• Multi-level analysis:

  • Integrate data from protein, mRNA, and functional levels

  • Combine in vitro and in vivo approaches

  • Utilize both genetic and pharmacological manipulation of NCF2/NADPH oxidase

• Temporal considerations:

  • Perform time-course studies to capture dynamic changes

  • Consider developmental stages and disease progression

  • Evaluate acute vs. chronic effects of NCF2 modulation

• Context-dependent mechanisms:

  • Research has shown that NCF2 deficiency has different outcomes depending on genetic background

  • On non-autoimmune backgrounds, NCF2 deficiency leads to infection susceptibility without autoimmunity

  • On autoimmune-prone backgrounds, even partial NCF2 deficiency accelerates autoimmunity

• Methodological triangulation:

  • Apply multiple techniques to measure the same parameter

  • Use complementary approaches (e.g., imaging, biochemical assays, and genetic models)

  • Collaborate with other labs to independently validate key findings

This multifaceted approach can help reconcile apparently contradictory findings about NCF2 function across different experimental systems and disease models .