CYBC1 Antibody

What is CYBC1 and why is it important in immunological research?

CYBC1 (cytochrome b-245 chaperone 1) is a 187 amino acid protein with a molecular weight of approximately 20.8 kDa that functions as a chaperone necessary for the stable expression of CYBA and CYBB subunits of the cytochrome b-245 heterodimer. This protein is highly expressed in immune cells, particularly macrophages, neutrophils, and monocytes, making it a crucial component of the innate immune response system. The CYBC1 gene has been directly associated with Chronic Granulomatous Disease (CGD), an immunodeficiency disorder characterized by recurrent bacterial and fungal infections. Understanding CYBC1's role is essential for elucidating the molecular mechanisms underlying CGD and potentially developing targeted therapeutics for immune disorders .

What are the common synonyms and alternative names for CYBC1?

When conducting literature searches or ordering reagents, researchers should be aware of multiple designations for CYBC1:

• Cytochrome b-245 chaperone 1

• C17orf62 (Chromosome 17 open reading frame 62)

• Eros (Essential for reactive oxygen species)

• Essential for reactive oxygen species protein

These alternative nomenclatures appear across different databases and commercial suppliers, so comprehensive searches should include all terms to ensure complete literature coverage.

What is the subcellular localization of CYBC1 and how does this impact experimental design?

CYBC1 is primarily localized to the endoplasmic reticulum (ER), which has significant implications for experimental design. When planning immunofluorescence or immunocytochemistry experiments, researchers should include appropriate ER markers (such as calnexin or PDI) for co-localization studies. Cell fractionation protocols should be optimized to efficiently isolate ER-enriched fractions when studying CYBC1. Additionally, fixation methods that preserve ER structure (such as paraformaldehyde fixation) are preferable for optimal visualization. When analyzing CYBC1 protein interactions within the ER microenvironment, techniques that maintain native protein conformations and interactions, such as proximity ligation assays or in situ proximity labeling methods, should be considered .

How do the different isoforms of CYBC1 affect antibody selection and experimental outcomes?

CYBC1 has two reported isoforms that can impact antibody recognition and experimental design. When selecting antibodies, researchers should carefully examine the epitope information to determine which isoform(s) the antibody recognizes. For comprehensive studies, consider using multiple antibodies targeting different regions to ensure detection of all relevant isoforms. Western blot analysis may reveal multiple bands corresponding to different isoforms, and researchers should validate these using positive controls with known isoform expression patterns.

For functional studies, isoform-specific knockdown or knockout approaches may be necessary to determine the distinct roles of each variant. When reporting results, clearly specify which isoform(s) were targeted to enable proper interpretation and reproducibility. A strategic approach might include:

• Initial screening with pan-CYBC1 antibodies

• Follow-up validation with isoform-specific antibodies

• Correlation of isoform expression with functional outcomes in your experimental system

What considerations should be made when studying CYBC1 across different species?

CYBC1 orthologs have been identified in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken. When designing cross-species studies, researchers should:

• Perform sequence alignments to identify conserved regions across species of interest

• Select antibodies raised against highly conserved epitopes for cross-reactivity

• Validate antibody specificity in each species through Western blot, using appropriate positive controls

• Consider potential differences in CYBC1 expression patterns, subcellular localization, and protein interactions across species

For evolutionary studies, leverage the conservation pattern of CYBC1 across vertebrates to investigate functional adaptation. When using animal models of granulomatous disease, account for species-specific variations in CYBC1 function that might affect disease presentation and progression .

How can machine learning approaches be integrated into CYBC1 antibody-antigen binding prediction?

Recent advances in computational biology offer promising avenues for predicting antibody-antigen interactions for CYBC1 research. Machine learning models can analyze many-to-many relationships between antibodies and antigens to predict binding specificity and affinity. When implementing these approaches:

• Start with library-on-library screening approaches to generate training data

• Apply active learning strategies to reduce experimental costs by starting with a small labeled subset and iteratively expanding based on model predictions

• Address out-of-distribution prediction challenges when test antibodies and antigens are not represented in training data

For CYBC1-specific applications, researchers should incorporate protein structural information and known binding epitopes as features in the model. A significant challenge is the cost of generating comprehensive experimental binding data, but active learning algorithms have been shown to reduce required experiments by up to 35% while accelerating the learning process. These computational approaches can guide epitope selection and antibody engineering for improved CYBC1 detection specificity .

What are the optimal applications for CYBC1 antibody detection, and how should protocols be optimized?

Based on current research practices, Western Blot, Immunocytochemistry (ICC), and Immunofluorescence (IF) are the most common and reliable applications for CYBC1 detection. For each application, specific optimizations are recommended:

Western Blot Optimization:

• Protein extraction: Use buffers containing mild detergents (0.5-1% NP-40 or Triton X-100) to efficiently extract CYBC1 from ER membranes

• Sample preparation: Heat samples at 70°C instead of boiling to prevent aggregation of this membrane-associated protein

• Blocking: 5% non-fat milk in TBST is typically effective, but for phospho-specific detection, switch to 5% BSA

• Antibody dilution: Start with 1:1000 dilution for most commercial antibodies, then optimize based on signal-to-noise ratio

• Detection: Enhanced chemiluminescence (ECL) systems provide adequate sensitivity for most applications

Immunofluorescence Optimization:

• Fixation: 4% paraformaldehyde for 15-20 minutes preserves CYBC1 localization

• Permeabilization: 0.1-0.2% Triton X-100 for 10 minutes typically provides sufficient access to ER-localized CYBC1

• Blocking: 5% normal serum (species different from primary antibody source) for 1 hour

• Co-staining: Include ER markers (calnexin, PDI) to confirm proper localization

• Controls: Include cells with CYBC1 knockdown as negative controls

What approaches are recommended for validating CYBC1 antibody specificity?

Thorough validation of CYBC1 antibody specificity is crucial for reliable research outcomes. A comprehensive validation strategy should include:

• Genetic approaches:

  • Compare staining in wild-type vs. CYBC1 knockout or knockdown cells

  • Use CRISPR-Cas9 edited cell lines with complete CYBC1 deletion

  • Employ siRNA or shRNA knockdown with recovery by exogenous expression

• Biochemical approaches:

  • Pre-absorption experiments with purified recombinant CYBC1 protein

  • Peptide competition assays using the immunizing peptide

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Multiple antibody approach:

  • Compare staining patterns using antibodies targeting distinct epitopes

  • Verify consistent localization and expression patterns across different antibodies

• Cross-validation with non-antibody methods:

  • Correlate protein detection with mRNA expression (RT-qPCR)

  • Use tagged CYBC1 constructs (GFP, FLAG) as secondary validation

Proper validation ensures that experimental observations are attributable to CYBC1 rather than non-specific interactions or artifacts .

How should researchers select between different conjugated forms of CYBC1 antibodies?

The selection of conjugated CYBC1 antibodies should be guided by the specific experimental application and technical requirements:

Biotin-Conjugated Antibodies:

Fluorophore-Conjugated Antibodies (e.g., FITC):

HRP-Conjugated Antibodies:

• Preferable for direct ELISA applications

• Eliminate potential cross-reactivity from secondary antibodies

• May offer improved sensitivity for low-abundance targets

The choice between conjugated forms should consider:

• Target abundance (lower abundance may benefit from signal amplification)

• Multiplexing requirements (direct conjugates can reduce species cross-reactivity issues)

• Downstream applications (certain conjugates are optimized for specific techniques)

• Background concerns (direct conjugates may reduce non-specific binding in certain tissues)

How should researchers interpret Western blot results for CYBC1?

When interpreting Western blot results for CYBC1, researchers should consider several key factors:

Expected banding pattern:

• The canonical form of human CYBC1 should appear at approximately 20.8 kDa

• Additional bands may represent post-translational modifications, different isoforms, or degradation products

• Verification of unexpected bands should be performed with multiple antibodies targeting different epitopes

Signal intensity interpretation:

• Quantification should be normalized to appropriate loading controls (β-actin, GAPDH, or preferably an ER-resident protein for compartment-specific normalization)

• Changes in CYBC1 expression should be correlated with functional outcomes related to cytochrome b-245 heterodimer formation and reactive oxygen species production

• Threshold for biological significance typically requires at least 1.5-2 fold changes with statistical validation across biological replicates

Common artifacts and their resolution:

• High molecular weight aggregates: Reduce sample heating temperature or modify detergent concentration

• Smeared bands: Improve protein extraction protocol or reduce protein loading

• Absence of signal: Confirm sample preparation preserves ER membrane proteins and validate antibody reactivity with positive controls

What are common pitfalls in CYBC1 research and how can they be avoided?

Researchers working with CYBC1 should be aware of several common challenges and implement strategies to overcome them:

Antibody cross-reactivity:

• Challenge: CYBC1 antibodies may cross-react with related proteins, particularly in non-human species

• Solution: Thoroughly validate antibodies using knockout controls and multiple antibodies targeting different epitopes

Cell type-specific expression:

• Challenge: CYBC1 expression varies significantly across cell types, with highest expression in macrophages, neutrophils, and monocytes

• Solution: Select appropriate positive control cell lines (e.g., THP-1, U937, or primary immune cells) and include negative controls with known low expression

Subcellular fractionation difficulties:

• Challenge: As an ER-localized protein, CYBC1 may be lost during standard cytosolic extraction protocols

• Solution: Use specialized ER extraction buffers and validate fractionation efficiency with established ER markers

Functional redundancy:

• Challenge: Compensatory mechanisms may mask phenotypes in CYBC1 knockdown/knockout studies

• Solution: Implement acute depletion strategies and comprehensive pathway analysis to detect subtle compensatory changes

Isoform-specific functions:

• Challenge: Different CYBC1 isoforms may have distinct functional roles

• Solution: Design isoform-specific targeting strategies and conduct parallel functional assays for each isoform

How can researchers effectively combine CYBC1 antibody studies with functional assays to understand its role in the NADPH oxidase complex?

To comprehensively investigate CYBC1's role in NADPH oxidase function, researchers should combine antibody-based detection with functional assays in an integrated workflow:

• Expression-function correlation:

  • Quantify CYBC1 levels via Western blot or immunofluorescence

  • Simultaneously measure ROS production using probes such as DHE, DCFDA, or luminol-enhanced chemiluminescence

  • Plot correlation between CYBC1 expression and functional output across multiple conditions or time points

• Interactome analysis:

  • Use co-immunoprecipitation with CYBC1 antibodies to pull down interaction partners

  • Perform Western blot analysis for known NADPH oxidase components (CYBA, CYBB, p47phox, etc.)

  • Consider proximity labeling approaches (BioID, APEX) to identify transient interactions

• Spatial-temporal dynamics:

  • Conduct live-cell imaging with fluorescently tagged CYBC1 constructs

  • Monitor co-localization with other NADPH oxidase components during cellular activation

  • Correlate changes in localization with ROS production in real-time

• Functional rescue experiments:

  • Deplete endogenous CYBC1 using siRNA or CRISPR-Cas9

  • Reintroduce wild-type or mutant CYBC1 variants

  • Assess restoration of NADPH oxidase assembly and function

This integrated approach provides mechanistic insights into how CYBC1 contributes to NADPH oxidase assembly, stability, and function, particularly in the context of immune cell activation and pathogen response .

How can active learning strategies improve antibody-antigen binding prediction for CYBC1 research?

Active learning represents a promising approach to optimize antibody-antigen binding prediction while minimizing experimental costs. The implementation process involves:

• Initial small-scale screening:

  • Begin with a limited subset of antibody-antigen pairs

  • Generate high-quality binding data for this initial training set

• Iterative model improvement:

  • Develop preliminary machine learning models based on initial data

  • Use model uncertainty to identify the most informative experiments to perform next

  • Iteratively expand the labeled dataset with strategically selected experiments

• Performance optimization:

  • Evaluate multiple active learning strategies (recent research evaluated fourteen novel strategies)

  • The most effective algorithms can reduce required experiments by up to 35%

  • Accelerate learning process by approximately 28 steps compared to random selection

• Application to CYBC1 research:

  • Map epitope specificity for various CYBC1 antibodies

  • Predict cross-reactivity with related proteins

  • Guide antibody engineering for improved specificity and affinity

This approach is particularly valuable for out-of-distribution prediction scenarios, where test antibodies and antigens differ from training data. Implementation of these methods can significantly reduce experimental costs while improving the accuracy of binding predictions for CYBC1 antibody research .

What is the current understanding of CYBC1's role in granulomatous disease pathophysiology?

CYBC1 has emerged as a critical factor in granulomatous disease, particularly through its role in regulating NADPH oxidase function:

• Molecular mechanism:

  • CYBC1 functions as a chaperone necessary for stable expression of CYBA and CYBB subunits

  • Deficiency leads to impaired assembly of functional NADPH oxidase complex

  • This results in decreased reactive oxygen species (ROS) production in phagocytes

• Clinical significance:

  • Mutations in CYBC1 have been identified in patients with chronic granulomatous disease (CGD)

  • These patients typically present with recurrent bacterial and fungal infections

  • CYBC1-associated CGD may have distinct clinical features compared to other genetic forms

• Diagnostic implications:

  • CYBC1 antibodies can be used for diagnostic immunoblotting to identify protein deficiency

  • Flow cytometry using CYBC1 antibodies may complement standard dihydrorhodamine (DHR) assays

  • Genetic testing should be considered in CGD patients with normal CYBB and CYBA genes

• Therapeutic considerations:

  • Gene therapy approaches targeting CYBC1 represent a potential treatment strategy

  • Small molecule enhancers of CYBC1 chaperone function could stabilize partially functional mutants

  • Understanding CYBC1 regulation may identify alternative therapeutic targets

Current research continues to elucidate the specific mechanisms by which CYBC1 mutations lead to disease and how these insights might inform novel therapeutic approaches .

How does CYBC1 expression vary across different immune cell subsets and activation states?

Understanding the differential expression of CYBC1 across immune cell populations provides valuable insight into its functional significance:

• Cell type-specific expression:

  • Highest expression observed in professional phagocytes: macrophages, neutrophils, and monocytes

  • Moderate expression in dendritic cells

  • Lower or absent expression in lymphoid cells (T cells, B cells, NK cells)

  • Expression correlates with cells that rely heavily on NADPH oxidase activity

• Regulation during immune activation:

  • CYBC1 expression may be upregulated during bacterial infection or exposure to PAMPs

  • IFN-γ stimulation can increase CYBC1 levels in monocytes and macrophages

  • Differentiation of monocytes to macrophages is accompanied by changes in CYBC1 expression

  • Activation-induced expression changes correlate with increased ROS production capacity

• Tissue-specific patterns:

  • Tissue-resident macrophages may exhibit distinct CYBC1 expression profiles

  • Microenvironmental factors can influence expression and function

  • Inflammatory conditions may alter expression patterns

• Methodological approaches:

  • Flow cytometry with permeabilization for intracellular CYBC1 staining

  • Immunohistochemistry of tissue sections to assess in situ expression

  • Single-cell RNA-seq to correlate CYBC1 expression with immune cell subtypes and states

These expression patterns provide important context for experimental design, suggesting appropriate cellular models and highlighting the physiological relevance of CYBC1 in specific immune cell functions .

What are the emerging trends in CYBC1 antibody applications for immunological research?

The field of CYBC1 antibody applications continues to evolve, with several emerging trends shaping future research directions:

• Multi-parameter analysis:

  • Integration of CYBC1 detection with other NADPH oxidase components in multiplexed assays

  • Correlation of CYBC1 expression with functional outputs (ROS production, phagocytosis, bacterial killing)

  • Single-cell analysis to reveal heterogeneity in CYBC1 expression and function

• Advanced imaging applications:

  • Super-resolution microscopy to visualize CYBC1 distribution within the ER at nanoscale resolution

  • Live-cell imaging with genetically encoded biosensors to correlate CYBC1 dynamics with ROS production

  • Intravital imaging to monitor CYBC1-dependent functions in immune cells during infection

• Clinical diagnostics:

  • Development of standardized CYBC1 antibody-based assays for CGD diagnosis

  • Correlation of CYBC1 expression levels with disease severity and prognosis

  • Potential biomarker applications in inflammatory and autoimmune conditions

• Therapeutic monitoring:

  • Assessing restoration of CYBC1 expression following gene therapy approaches

  • Monitoring CYBC1 levels during immunomodulatory treatments

  • Development of companion diagnostics for emerging CGD therapies

These trends highlight the importance of continuing to develop and validate high-quality CYBC1 antibodies for diverse research applications .

How can researchers contribute to standardizing CYBC1 antibody validation and reporting?

Standardization of CYBC1 antibody validation and reporting is essential for research reproducibility. Researchers can contribute through several approaches:

• Comprehensive validation reporting:

  • Document validation methods in publications (knockout controls, multiple antibodies, etc.)

  • Specify exact catalog numbers, lot numbers, and dilutions used

  • Include representative images of positive and negative controls

• Antibody registry participation:

  • Register validated antibodies in community databases (e.g., Antibodypedia, CiteAb)

  • Contribute validation data to repositories like the Antibody Registry

  • Assign Research Resource Identifiers (RRIDs) to antibodies in publications

• Methodological transparency:

  • Share detailed protocols through platforms like protocols.io

  • Specify critical parameters that affect antibody performance

  • Report both successful and failed antibody applications

• Collaborative validation initiatives:

  • Participate in multi-laboratory validation studies

  • Contribute to consensus guidelines for CYBC1 detection methods

  • Share reference materials and standards with the research community