CYBRD1 Antibody

What is CYBRD1 and what is its physiological function?

CYBRD1, also known as duodenal cytochrome b (Dcytb), is a putative plasma membrane diheme protein that functions in iron absorption. It is primarily induced in mouse duodenal mucosa under conditions requiring accelerated intestinal iron absorption, including iron deficiency, hypoxia, hypotransferrinemia, pregnancy, and hemolytic anemia . CYBRD1 is situated on the brush-border membrane of mature duodenal enterocytes and confers ferric reductase activity when expressed in experimental systems such as Xenopus oocytes or cultured mammalian cells .

What is the molecular structure and cellular distribution of CYBRD1?

CYBRD1 typically appears on immunoblots as two specific bands: one at 30-35 kDa (consistent with its predicted monomeric mass) and another at 60-70 kDa (potentially representing a dimer that persists under standard denaturing conditions) . The murine CYBRD1 gene consists of 4 exons on chromosome 2, with exon 2 encoding the putative binding sites for cofactors essential for its reductase activity . The protein localizes primarily to the duodenal brush border membrane, with expression patterns varying according to iron status.

What types of CYBRD1 antibodies are available for research applications?

Multiple CYBRD1 antibodies are commercially available with varying characteristics:

• Antibodies targeting different epitopes: AA 51-150, AA 220-286, AA 215-286, and C-terminal regions

• Host species: Primarily rabbit and goat polyclonal antibodies

• Conjugation options: Unconjugated, HRP-conjugated, FITC-conjugated, and biotin-conjugated variants

• Species reactivity: Antibodies specifically validated for human, mouse, and rat CYBRD1

What critical validation steps should researchers perform when using CYBRD1 antibodies?

Comprehensive validation should include:

• Positive and negative controls using tissues with known CYBRD1 expression patterns (high in duodenum, variable in other tissues)

• Comparison of wild-type versus knockout samples when available

• Peptide competition assays to confirm specificity

• Verification of expected molecular weight patterns (30-35 kDa monomer and potential 60-70 kDa dimer)

• Cross-validation across multiple applications (WB, IHC, IF) to ensure consistent detection patterns

Which applications are most suitable for CYBRD1 antibody use?

CYBRD1 antibodies have been successfully employed in multiple experimental approaches:

• Western Blotting (WB): Effective for quantifying expression levels and detecting specific molecular weight forms

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for high-throughput quantitative analysis

• Immunohistochemistry (IHC): On both paraffin-embedded and frozen sections for localization studies

• Immunofluorescence (IF): On both cultured cells and tissue sections for co-localization studies with other proteins

How can CYBRD1 antibodies be effectively utilized in cancer research?

Recent studies have demonstrated CYBRD1's potential role in cancer progression, particularly in gliomas. Methodological approaches include:

• Expression analysis in tumor grading: Using IHC to correlate CYBRD1 expression with tumor grade. Studies have shown that CYBRD1-positive cells increase with glioma grade, with WHO IV samples exhibiting the highest expression .

• Functional validation studies: Combining antibody-based detection with genetic manipulation (overexpression or silencing) to establish causality. In glioma LN229 and T98G cell lines, CYBRD1 overexpression promoted cell viability, migration, and invasion, while silencing attenuated these aggressive characteristics .

• Prognostic marker development: CYBRD1 has been identified as a potential risk factor in glioma recurrence, with significant upregulation in tumor tissues compared to non-tumor samples .

What techniques are most effective for CYBRD1 detection in tissue samples?

For optimal CYBRD1 detection in tissues:

• Sample preparation: Fix tissues in 4% paraformaldehyde, embed in paraffin, and section to 4-μm thickness .

• Immunostaining protocol:

  • Deparaffinize and rehydrate sections

  • Perform antigen retrieval (typically heat-induced with citrate buffer)

  • Block with 5% BSA for 2 hours at room temperature

  • Incubate with primary anti-CYBRD1 antibody overnight at 4°C

  • Apply appropriate secondary antibody and detection system

  • Counterstain and mount

• Controls: Include both positive controls (duodenal tissue) and negative controls (either antibody omission or tissues known to lack CYBRD1 expression).

What are the critical parameters for Western blot detection of CYBRD1?

For reliable Western blot results:

• Protein extraction: Use RIPA buffer with protease inhibitors to efficiently extract CYBRD1 from membrane fractions .

• Sample preparation:

  • Quantify protein using BCA assay

  • Load 20 μg protein per lane

  • Separate by SDS-PAGE (4-12% gradient gels work well)

• Transfer conditions:

  • Transfer to PVDF membrane

  • Block with 5% BSA for 2 hours at room temperature

• Antibody incubation:

  • Incubate with primary anti-CYBRD1 antibody overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody

  • Develop using enhanced chemiluminescence

• Expected results: Look for specific bands at 30-35 kDa (monomer) and potentially 60-70 kDa (possible dimer) .

How can researchers address inconsistencies between CYBRD1 protein and mRNA expression?

When confronting discrepancies between protein and transcript levels:

• Temporal considerations: mRNA expression often precedes protein upregulation; consider time-course experiments.

• Post-transcriptional regulation: Assess microRNA regulation or RNA stability factors that might affect translation efficiency.

• Methodological validation:

  • Confirm antibody specificity through multiple controls

  • Validate RNA extraction and RT-qPCR protocols (use GAPDH or other appropriate housekeeping genes)

  • Consider absolute quantification methods for more accurate comparisons

• Functional validation: Complement expression studies with functional assays (e.g., ferric reductase activity) to evaluate biological significance of expression changes.

What insights do CYBRD1 knockout models provide for understanding iron metabolism?

Studies with CYBRD1 knockout models have yielded surprising findings:

• Phenotypic observation: CYBRD1-/- mice show little, if any, effect on body iron accumulation, even when maintained on iron-deficient diets .

• Gene expression patterns: Expression of other key iron metabolism genes (SLC11A2, SLC40A1, HAMP) remains unchanged in CYBRD1-/- mice compared to wild-type mice on iron-deficient diets .

• Experimental validation: These findings were confirmed through:

  • Serum iron assays

  • Nonheme liver iron concentration measurements

  • Northern blot analysis of iron regulatory genes

  • Immunoblot analysis of duodenal enterocyte lysates

• Research implications: These results suggest:

  • Potential redundancy in intestinal iron reduction mechanisms

  • The existence of alternative pathways for dietary iron absorption

  • The need to reconsider the relative importance of CYBRD1 in iron metabolism

How do research findings on CYBRD1 in glioma inform experimental design for other cancers?

The association between CYBRD1 and glioma progression provides methodological frameworks for studying other cancers:

• Gene expression screening: CYBRD1 was identified as a risk factor with hazard ratio >1.5, highlighting the value of comprehensive expression profiling and survival analysis in identifying novel cancer-associated genes .

• Validation pipeline:

  • Initial screening in patient samples (comparing normal vs. tumor tissues)

  • Correlation with clinical parameters (grade, stage, survival)

  • Functional validation through overexpression and silencing experiments

  • Phenotypic assessment (viability, migration, invasion assays)

• Mechanistic investigation: Future studies should explore how CYBRD1-related iron metabolism may contribute to cancer progression through:

  • Effects on oxidative stress

  • Modulation of iron-dependent enzymes

  • Potential interactions with established oncogenic pathways

What novel applications of CYBRD1 antibodies are emerging in research?

Emerging applications include:

• Single-cell protein expression profiling: Using CYBRD1 antibodies for mass cytometry or imaging mass cytometry to characterize cellular heterogeneity in tissues.

• Protein-protein interaction studies: Employing co-immunoprecipitation with CYBRD1 antibodies to identify novel binding partners in different cellular contexts.

• High-resolution localization: Utilizing super-resolution microscopy with CYBRD1 antibodies to precisely map its distribution within membrane microdomains.

• Therapeutic target validation: Using antibodies to evaluate CYBRD1 as a potential therapeutic target, particularly in cancers where it shows aberrant expression.

How might contradictory findings about CYBRD1 function be reconciled through improved antibody-based methodologies?

To address conflicting results in the literature:

• Standardized methodologies: Develop consensus protocols for CYBRD1 detection across different experimental systems.

• Comprehensive antibody validation:

  • Validate antibodies against knockout controls

  • Compare results from antibodies targeting different epitopes

  • Establish clear criteria for positive/negative staining

• Context-specific analysis: Systematically evaluate CYBRD1 function across:

  • Different tissues and cell types

  • Various physiological and pathological conditions

  • Developmental stages

  • Genetic backgrounds

• Integration with emerging technologies: Combine antibody-based detection with proteomics, transcriptomics, and functional genomics approaches to build a more complete understanding of CYBRD1 biology.