FANCA Antibody, Biotin conjugated

What is FANCA and why is it a significant research target?

FANCA, also known as FAA, FACA, and FANCH, is a 163 kDa DNA repair protein that plays a crucial role in postreplication repair and cell cycle checkpoint functions. It forms part of the eight-subunit Fanconi anemia core complex that ubiquitinates FANCD2 and FANCI, contributing to the reactivation of stalled DNA repair replication forks . FANCA is widely expressed, activated by phosphorylation, and partners with FANCG. Recent research has demonstrated elevated FANCA expression in various cancers, including gastric cancer, making it an important target for oncology research .

What are the common applications for biotinylated FANCA antibodies?

Biotinylated FANCA antibodies are versatile research tools with multiple applications:

The biotin conjugation facilitates detection through the strong biotin-streptavidin interaction (kd of 4 × 10^-14 M), allowing for signal amplification and versatile detection strategies .

How should biotinylated FANCA antibodies be stored for optimal stability?

Proper storage is crucial for maintaining antibody functionality. Biotinylated FANCA antibodies should typically be stored at -20°C for up to 12 months . Most commercial preparations come in an aqueous buffered solution containing TBS (pH 7.4) with BSA, preservatives like Proclin300, and 50% glycerol . To minimize degradation, follow these research-validated practices:

• Aliquot upon receipt to avoid repeated freeze-thaw cycles

• Store in a manual defrost freezer to prevent temperature fluctuations

• When refrigerated (2-8°C), limit storage to approximately one month under sterile conditions

• Return to -20°C to -70°C for long-term storage of 6 months or longer

What are the optimal biotinylation methods for FANCA antibodies?

Researchers have compared different biotinylation techniques, with significant differences in performance. Two primary methods stand out:

• ZBPA Domain Method: This approach utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc portion of antibodies. Research demonstrates that ZBPA biotinylation results in distinct immunoreactivity without off-target staining, regardless of stabilizing proteins in the buffer . This method shows superior specificity for in situ protein detection in tissues.

• Commercial Kits (e.g., Lightning-Link): While convenient, commercial kits may not specifically target IgG and could potentially label stabilizing proteins present in antibody solutions. Studies show that these methods can sometimes result in characteristic patterns of nonspecific staining .

For sensitive applications like immunohistochemistry, the ZBPA method is recommended as it provides a more stringent approach to antibody biotinylation .

How can I optimize immunohistochemistry protocols using biotinylated FANCA antibodies?

Successful immunohistochemistry with biotinylated FANCA antibodies requires attention to multiple technical factors:

• Antigen Retrieval: Use TE buffer at pH 9.0 for optimal results. Alternatively, citrate buffer at pH 6.0 may be used, but comparative studies suggest TE buffer provides better epitope exposure .

• Tissue Processing Protocol:

  • Deparaffinize tissue sections with xylene

  • Rehydrate using graded alcohol solutions and citrate buffer

  • Block with 3% hydrogen peroxide

  • Incubate with primary antibody (typical dilution 1:100 for FANCA)

  • Apply biotin-conjugated secondary antibody

  • Incubate with streptavidin-peroxidase complex

• Documentation: For quantitative analysis, select five high-power fields (400× magnification) randomly from each slide. Score protein expression based on both the proportion of positive cells and staining intensity .

What controls should be included when using biotinylated FANCA antibodies?

Rigorous experimental design requires appropriate controls:

• Positive Tissue Controls: Based on validated research, include:

  • Human colon cancer tissue

  • Human cervical cancer tissue

  • Human stomach cancer tissue

  • Mouse intestine or thymus tissue

  • Rat small intestine or testis tissue

• Positive Cell Line Controls: Western blot analyses have validated:

  • COLO 320 cells

  • HeLa cells

  • MOLT-4 human acute lymphoblastic leukemia cell line

• Negative Controls: Include:

  • Primary antibody omission

  • Isotype-matched irrelevant antibody

  • Tissues known to be negative for FANCA

Including these controls ensures the specificity of staining patterns and helps distinguish true signal from background.

How can biotinylated FANCA antibodies be used to investigate FANCA's role in cancer?

Recent research has revealed significant correlations between FANCA expression and cancer progression. A 2024 study demonstrated that FANCA expression is distinctly elevated in gastric cancer cells compared to normal tissues . Biotinylated FANCA antibodies can be employed to:

• Evaluate Clinical Correlations: Investigate associations between FANCA expression and clinicopathological characteristics. Research has shown correlations with:

  • Tumor size (P=0.003)

  • Depth of invasion (P=0.049)

  • Lymph node metastasis (P=0.007)

  • Distant metastasis (P=0.026)

  • TNM stage

• Compare Expression Across Cancer Types: Validated research protocols have confirmed FANCA antibody reactivity in multiple cancer tissues, enabling comparative studies of expression patterns in:

  • Colon cancer

  • Cervical cancer

  • Stomach cancer

  • Head and neck squamous cell carcinoma

• Investigate Cell Cycle Pathway Interactions: Utilize Gene Set Enrichment Analysis (GSEA) to explore associations between FANCA expression and cell cycle-related genes, as demonstrated in recent gastric cancer research .

What approaches can resolve discrepancies in FANCA detection across different assays?

When researchers encounter inconsistent results with biotinylated FANCA antibodies, several methodological approaches can help resolve these discrepancies:

• Epitope Mapping: Different antibodies may target distinct epitopes of FANCA (full length is 1455 amino acids). Compare antibody immunogens:

  • Some target specific regions (e.g., Tyr35-Arg142)

  • Others use KLH-conjugated synthetic peptides

  • Check if your target region contains known mutations or splice variants

• Cross-Validation with Multiple Detection Methods:

  • Combine biotinylated antibody detection with non-biotinylated systems

  • Verify results using genetic approaches (siRNA knockdown or CRISPR knockout)

  • Compare results with computational predictions from databases like TCGA

• Sample-Specific Optimization: As noted in technical documentation, optimal conditions can be "sample-dependent." Perform titration experiments with each new tissue or cell type to determine optimal antibody concentration .

How can biotinylated FANCA antibodies contribute to understanding DNA repair mechanisms?

FANCA is crucial for maintaining genomic stability through its role in the Fanconi anemia pathway. Biotinylated FANCA antibodies enable detailed investigations of:

• Protein Complex Formation: Using immunoprecipitation, researchers can isolate FANCA-containing complexes to:

  • Identify interaction partners beyond known associations (e.g., FANCG)

  • Characterize the eight-subunit Fanconi anemia core complex formation

  • Study post-translational modifications that regulate complex assembly

• Subcellular Localization During DNA Damage Response: Through immunofluorescence, track FANCA movement between:

  • Cytoplasm (baseline location)

  • Nucleus (during DNA damage response)

  • Nuclear foci (sites of active DNA repair)

• Temporal Dynamics: Investigate the kinetics of FANCA recruitment to DNA damage sites and its correlation with cell cycle phases, particularly at the G1/S transition where recent research has indicated important functional roles .

What strategies can address high background when using biotinylated FANCA antibodies?

High background is a common challenge with biotinylated antibodies due to endogenous biotin in tissues. Research-validated approaches to minimize this include:

• Endogenous Biotin Blocking: Prior to antibody application, block endogenous biotin using:

  • Avidin-biotin blocking kit

  • Streptavidin/biotin blocking reagents

  • 0.1% sodium borohydride treatment

• Biotinylation Method Consideration: Studies have demonstrated that ZBPA-based biotinylation produces significantly less off-target staining compared to other methods. As reported in comparative research: "For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining."

• Modified Detection System: Consider using:

  • Polymer-based detection systems

  • Tyramide signal amplification for increased sensitivity at lower antibody concentrations

  • Multi-step avidin-biotin complex (ABC) methods with additional washing steps

How can I validate specificity when using biotinylated FANCA antibodies?

Rigorous validation is essential for reliable research results. Implement these methodological approaches:

• Genetic Validation: Use FANCA knockout or knockdown models:

  • siRNA or shRNA targeting FANCA

  • CRISPR/Cas9-mediated FANCA knockout cell lines

  • Compare staining patterns between wild-type and FANCA-depleted samples

• Peptide Competition Assays: Pre-incubate the antibody with excess immunizing peptide before application to samples. Specific staining should be abolished or significantly reduced.

• Cross-Validation with Multiple Antibodies: Compare results using:

  • Different commercial anti-FANCA antibodies (e.g., Proteintech 11975-1-AP, Bethyl A301-980A)

  • Antibodies targeting different epitopes of FANCA

  • Alternative detection methods (direct vs. indirect)

What are the critical parameters for optimizing Western blot detection of FANCA using biotinylated antibodies?

FANCA is a high molecular weight protein (163 kDa), presenting specific challenges for Western blot detection. Research-optimized protocols suggest:

• Sample Preparation Considerations:

  • Use freshly prepared protein lysates when possible

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors to preserve phosphorylated forms of FANCA

• Gel Electrophoresis Parameters:

  • Use low percentage gels (6-8%) for better resolution of high molecular weight proteins

  • Extend transfer time for complete migration of large proteins

  • Consider wet transfer for more efficient movement of large proteins

• Detection Optimization:

  • Typical Western blot dilution ranges from 1:1000-1:5000 for primary antibody

  • For biotinylated antibodies, use streptavidin-HRP at 1:2000-1:5000

  • Longer exposure times may be necessary for optimal visualization

  • Use reducing conditions as demonstrated in validated protocols

Published research has confirmed successful FANCA detection in MOLT-4 human acute lymphoblastic leukemia cell line, showing a specific band at approximately 160 kDa under reducing conditions .

How can biotinylated FANCA antibodies contribute to cancer biomarker development?

Emerging research highlights FANCA's potential as a cancer biomarker. Biotinylated FANCA antibodies facilitate this research through:

• Tissue Microarray (TMA) Analysis: Recent studies employed TMAs comprising 80 patients with histologically confirmed gastric cancer and 80 control subjects to evaluate FANCA expression patterns . Biotinylated antibodies enable:

  • High-throughput screening across multiple tissue samples

  • Standardized staining conditions for comparative analysis

  • Quantitative assessment of expression levels

• Correlation with Genomic Data: Integrate antibody-based protein detection with:

  • Gene expression data from TCGA

  • Mutation profiles from next-generation sequencing

  • Copy number variation analysis

• Multi-marker Panels: Combine FANCA detection with other Fanconi anemia pathway components for comprehensive pathway analysis in cancer tissues.

What are the considerations for multiplexed detection systems incorporating biotinylated FANCA antibodies?

Advanced research frequently requires simultaneous detection of multiple proteins. When incorporating biotinylated FANCA antibodies into multiplexed systems:

• Compatible Fluorophore Selection: When combining with streptavidin-conjugated fluorophores:

  • Choose fluorophores with minimal spectral overlap

  • Consider quantum dots for narrow emission spectra

  • Validate signal separation with single-color controls

• Sequential Detection Protocols: For co-localization studies:

  • Begin with the least abundant target (often FANCA)

  • Include stringent washing steps between detection rounds

  • Consider tyramide signal amplification for weak signals

• Cross-Reactivity Assessment: Validate that:

  • Streptavidin conjugates do not bind non-specifically to other primary antibodies

  • Secondary detection systems do not recognize the biotinylated FANCA antibody

  • Controls with individual omission of each component confirm specificity

How can computational approaches enhance analysis of data generated with biotinylated FANCA antibodies?

Modern research integrates wet-lab experiments with computational analysis. For FANCA studies:

• Pathway Analysis Integration: Recent research has employed:

  • StringDB to identify FANCA-interacting genes

  • KEGG and GO enrichment analysis using DAVID bioinformatics tools

  • Analysis criteria including minimum gene set of 5, maximum gene set of 5000, with significance defined by P<0.05 and FDR<0.25

• Quantitative Image Analysis:

  • Automated scoring systems for immunohistochemistry

  • Machine learning approaches for pattern recognition

  • Correlation of staining patterns with clinical outcomes

• Multi-omics Data Integration:

  • Correlate protein expression (detected via biotinylated antibodies) with:

  • Transcriptomics data (RNA-seq)

  • Genomic alterations (mutations, CNVs)

  • Epigenetic modifications

This comprehensive approach has revealed that FANCA is not just a DNA repair protein but also plays significant roles in cell cycle progression, particularly at the G1/S transition, with implications for cancer development and therapy resistance .