C19orf47 Antibody

What is C19orf47 and why are antibodies against it important for research?

C19orf47 (Chromosome 19 Open Reading Frame 47) is a protein-coding gene that produces an uncharacterized protein. While its precise function remains under investigation, antibodies against C19orf47 serve as crucial tools for detecting and characterizing this protein in various experimental contexts. The protein has the UniProt ID Q8N9M1 and is also known by alternative names such as FLJ36888 .

These antibodies enable researchers to:

• Detect C19orf47 protein expression in different tissues and cell lines

• Study the protein's subcellular localization

• Investigate potential interactions with other proteins

• Analyze expression changes in various physiological and pathological conditions

Current research suggests C19orf47 functions as part of a larger protein complex that interacts with key cellular machinery to modulate various biological processes .

What types of C19orf47 antibodies are currently available for research?

Most commercially available C19orf47 antibodies are rabbit polyclonal antibodies raised against different immunogen sequences. For example:

• HPA046309 uses the sequence AWDSDNDSSSSVLQYAGVLKKLGRGPAKASPQPALTVKAKATSSATTAAAPTLRRLALSSRSGLERKPESLSKVSIIK

• PA5-61210 uses a similar N-terminal sequence

• PA5-59751 uses a different sequence: TVVGDIIAILKHAKVVHRQDMCKAATESVPCSPSPLAGEIRRGTSAASRMITNSLNHDSPPSTPPRRPDTSTSKISVT

The diversity of immunogens provides researchers with options to target different epitopes of the C19orf47 protein.

What applications are C19orf47 antibodies validated for?

Most C19orf47 antibodies have been validated for multiple applications, though performance may vary between manufacturers and specific antibodies:

When selecting an antibody, researchers should consider the specific validation data for their intended application. For example, an antibody that performs well in Western blot may not necessarily perform equally well in immunofluorescence applications .

How should I validate a C19orf47 antibody before using it in my research?

Antibody validation is crucial for ensuring reliable experimental results. Based on current best practices, C19orf47 antibody validation should follow a systematic approach:

• Genetic Approach (Gold Standard):

  • Generate CRISPR/Cas9 knockout (KO) cell lines for C19orf47 in a cell line with high endogenous expression

  • Compare antibody signals between parental and KO cell lines

  • A specific antibody should show signal in parental cells but not in KO cells

• Orthogonal Approach:

  • Compare antibody detection with an independent method (e.g., mass spectrometry)

  • Correlate RNA expression data with protein detection

  • While useful for Western blot validation, this approach is less reliable for immunofluorescence

• Multiple Antibody Approach:

  • Test different antibodies targeting different epitopes of C19orf47

  • Consistent localization or detection patterns across antibodies increase confidence

• Application-Specific Validation:

  • For Western blot: Verify correct molecular weight (predicted ~45 kDa for C19orf47)

  • For IHC/IF: Include appropriate positive and negative control tissues

  • For IP: Confirm enrichment compared to starting material

Remember that validation in one application doesn't guarantee performance in another. Research indicates that genetic approaches using KO cells provide the most robust validation, with 80-89% of antibodies validated by genetic strategies showing confirmed performance across applications .

What are the optimal experimental conditions for Western blot detection of C19orf47?

Successful Western blot detection of C19orf47 requires optimization of several parameters:

Sample Preparation:

• Use tissues/cells with confirmed C19orf47 expression (human testis, small intestine, and muscle tissues show detectable expression)

• For cell lines, HEK293 cells have been used successfully for C19orf47 detection

• Use a complete lysis buffer containing protease inhibitors

Protocol Optimization:

• Protein Loading: 20-50 μg total protein per lane

• Transfer: Standard wet transfer protocols are suitable

• Blocking: 5% non-fat milk or BSA in TBST (1 hour, room temperature)

• Primary Antibody: Dilutions typically range from 1:500 to 1:2000 (optimize for each antibody)

• Incubation: Overnight at 4°C provides optimal results

• Secondary Antibody: Anti-rabbit HRP (most C19orf47 antibodies are rabbit-derived)

• Detection: Both chemiluminescence and fluorescence detection methods are suitable

Troubleshooting Tips:

• If no signal is detected, consider concentrating the sample or using a cell line with higher C19orf47 expression

• Multiple bands may indicate splice variants, post-translational modifications, or non-specific binding

• Always include positive controls (e.g., recombinant C19orf47 protein) and negative controls (e.g., KO cell lysate if available)

The predicted molecular weight of C19orf47 is approximately 45 kDa, but variations may occur due to post-translational modifications .

What controls should be included when using C19orf47 antibodies for immunohistochemistry?

Robust controls are essential for reliable immunohistochemistry experiments with C19orf47 antibodies:

Positive Controls:

• Human testis tissue has shown positive staining with C19orf47 antibodies

• Human small intestine tissue also demonstrates detectable C19orf47 expression

• Use tissues known to express the target protein based on RNA-seq or proteomics data

Negative Controls:

• Primary antibody omission (to detect potential secondary antibody non-specific binding)

• Isotype control antibodies (same species and isotype as the primary antibody)

• Ideally, tissues from C19orf47 knockout models, though these may not be readily available

• Pre-absorption with immunizing peptide (if available from manufacturer)

Procedural Controls:

• Standardize fixation methods (preferably formalin-fixed, paraffin-embedded)

• Optimize antigen retrieval methods (heat-induced epitope retrieval may be necessary)

• Titrate antibody concentrations (typically 1:50-1:200 dilutions for IHC)

• Include both positive and negative cell types within the same section when possible

Interpretation Guidelines:

• Document subcellular localization patterns

• Assess staining intensity using standardized scoring systems

• Compare staining patterns with published literature when available

• Consider dual labeling with markers of specific subcellular compartments to confirm localization

How can I use C19orf47 antibodies for immunoprecipitation studies?

Immunoprecipitation (IP) with C19orf47 antibodies requires careful optimization:

Protocol Recommendations:

• Antibody Selection:

  • Not all C19orf47 antibodies work effectively for IP

  • Test multiple antibodies if possible, as IP efficiency varies significantly between antibodies

• Sample Preparation:

  • Use cell lines with confirmed high C19orf47 expression

  • For detergent selection, start with a mild non-ionic detergent (e.g., 1% NP-40 or 1% Triton X-100)

  • Include protease and phosphatase inhibitors in lysis buffers

• IP Procedure:

  • Pre-clear lysates to reduce non-specific binding

  • Pre-couple antibodies to protein A/G beads (2-5 μg antibody per IP reaction)

  • Incubate overnight at 4°C with gentle rotation

  • Wash stringently (at least 4-5 washes) to reduce background

• Controls:

  • Input control (5-10% of starting material)

  • Beads-only control (no antibody)

  • IgG isotype control

  • Ideally, C19orf47 knockout cell lysate as negative control

• Detection:

  • Western blot using a different C19orf47 antibody raised in another species

  • If using rabbit antibodies for IP, detect with mouse anti-C19orf47 (or vice versa)

Based on studies with other proteins, optimizing antibody-bead coupling conditions can significantly improve IP efficiency . For C19orf47 IP specifically, comparing enrichment to starting material helps evaluate antibody performance.

What approaches can be used to investigate potential C19orf47 protein interactions?

Investigating C19orf47 protein interactions requires multiple complementary approaches:

Co-Immunoprecipitation (Co-IP):

• Perform IP with C19orf47 antibodies following the protocol in section 3.1

• Analyze co-precipitated proteins by:

  • Western blot for specific suspected interactors

  • Mass spectrometry for unbiased discovery of interacting partners

• Validate interactions by reverse Co-IP (use antibodies against the interacting protein)

Proximity Labeling:

• Generate C19orf47 fusion constructs with enzymes like BioID or APEX2

• Express in relevant cell types to biotinylate proteins in close proximity

• Purify biotinylated proteins and identify by mass spectrometry

• Validate top hits using complementary methods

Protein-Protein Interaction Screens:

• Yeast two-hybrid screening with C19orf47 as bait

• Mammalian two-hybrid or split luciferase assays for candidate validation

• In vitro binding assays with recombinant proteins

Bioinformatic Predictions:

• Use interaction databases (STRING, BioGRID) to identify potential interactors

• Perform domain-based predictions for potential binding partners

• Analyze co-expression data across tissues/conditions

Visualization Approaches:

• Fluorescence Resonance Energy Transfer (FRET)

• Proximity Ligation Assay (PLA) using C19orf47 antibodies and antibodies against candidate interactors

• Co-localization studies using confocal microscopy

Remember to include appropriate controls and validate interactions through multiple orthogonal methods, as protein interaction studies are prone to false positives.

How can cross-reactivity issues with C19orf47 antibodies be addressed in multi-species studies?

When using C19orf47 antibodies across multiple species, addressing cross-reactivity is crucial:

Sequence Homology Analysis:

• Human C19orf47 shows relatively high sequence homology with mouse (83-87%) and rat (85-86%) orthologs

• This suggests potential cross-reactivity, but experimental validation is essential

Cross-Reactivity Testing Strategy:

• In silico Analysis:

  • Compare immunogen sequences with orthologs across species

  • Identify regions of high conservation vs. divergence

  • Some antibodies report specific homology percentages (e.g., PA5-61210 reports 83% homology with mouse)

• Experimental Validation:

  • Test antibodies on samples from each species of interest

  • Use species-specific positive controls (tissues known to express C19orf47)

  • Include knockout controls from each species when possible

  • Compare staining patterns across species for consistency

• Specialized Approaches:

  • Pre-absorption with species-specific peptides to reduce non-specific binding

  • Generation of species-specific antibodies if cross-reactivity is problematic

  • Epitope mapping to identify species-specific regions

Interpretation Considerations:

• Different antibody dilutions may be required for optimal results across species

• Subcellular localization may vary between species due to evolutionary differences

• Expression patterns may differ between orthologs

• Western blot may show different band patterns or molecular weights between species

For C19orf47 specifically, while some antibodies claim cross-reactivity with mouse and human , researchers should validate this experimentally for their specific applications.

What factors contribute to batch-to-batch variability in C19orf47 antibody performance?

Batch-to-batch variability is a significant concern for antibody-based research, particularly with polyclonal antibodies:

Sources of Variability:

• Antibody Production Factors:

  • Polyclonal antibodies (like most C19orf47 antibodies) inherently show more variability than monoclonals

  • Different animals used for immunization

  • Variations in immunization protocols

  • Differences in purification methods

  • Storage and handling during production

• Quality Control Issues:

  • Variations in validation methods between batches

  • Different thresholds for passing quality control

  • Inconsistent testing across applications

• Storage and Handling:

  • Freeze-thaw cycles

  • Improper temperature storage

  • Buffer composition changes

  • Microbial contamination

Mitigation Strategies:

• Documentation:

  • Record lot numbers for all experiments

  • Maintain detailed protocols and results for each antibody lot

  • Archive aliquots of well-performing lots for critical experiments

• Validation:

  • Re-validate each new lot using knockout controls if possible

  • Compare directly with previous lots in side-by-side experiments

  • Test across all intended applications with each new lot

• Alternative Approaches:

  • Consider recombinant antibodies for long-term projects

  • Use multiple antibodies targeting different epitopes

  • Maintain frozen aliquots of validated lots to minimize freeze-thaw cycles

Research has shown that even with standardized production processes, polyclonal antibodies can show significant variability that impacts experimental reproducibility . For critical experiments, researchers should validate each new lot and consider renewable antibody sources.

How can I troubleshoot non-specific binding issues with C19orf47 antibodies?

Non-specific binding is a common challenge when working with antibodies, including those against C19orf47:

Common Causes of Non-Specific Binding:

• Antibody-Related Factors:

  • Cross-reactivity with similar epitopes on unrelated proteins

  • Fc receptor binding

  • High antibody concentration

  • Low affinity or poor specificity

• Protocol-Related Factors:

  • Insufficient blocking

  • Inappropriate blocking agent

  • Inadequate washing

  • Sample overloading (Western blot)

  • Over-fixation (IHC/IF)

Troubleshooting Approaches:

Verification Strategies:

• Perform peptide competition assays

• Use knockout or knockdown controls

• Test specificity with pre-immune serum (for polyclonal antibodies)

• Perform immunodepletion experiments

• Compare staining patterns across multiple antibodies against different epitopes

The gold standard for addressing non-specific binding remains genetic approaches using knockout controls, which have been shown to be most effective for determining antibody specificity .

What technological advances are improving C19orf47 antibody specificity and reproducibility?

Recent technological advances are addressing long-standing challenges in antibody research:

Emerging Technologies:

• Recombinant Antibody Production:

  • Single-cell sequencing of B cells to capture antibody sequences

  • Expression of antibodies in controlled systems rather than animals

  • Elimination of batch-to-batch variability through defined sequences

  • While not yet widely available for C19orf47, recombinant antibodies represent the future of reproducible research

• CRISPR-Based Validation:

  • Generation of knockout cell lines for definitive antibody validation

  • Development of cell libraries with multiple gene knockouts

  • Creation of epitope-tagged endogenous proteins for validation

  • Standardized procedures for antibody testing against knockout controls

• Improved Screening Methods:

  • High-throughput protein arrays for cross-reactivity testing

  • Peptide scanning for precise epitope mapping

  • Structural analysis of antibody-antigen interactions

  • Machine learning algorithms to predict antibody specificity

• Standardization Initiatives:

  • Development of community standards for antibody validation

  • Creation of shared validation resources (e.g., knockout cell banks)

  • Implementation of minimum reporting standards for antibody experiments

  • Antibody validation databases with standardized metrics

Application to C19orf47 Research:

• Several initiatives are working to create standardized validation procedures across antibodies

• Enhanced validation protocols using genetic approaches provide more reliable C19orf47 antibodies

• Open science platforms are improving sharing of antibody validation data

• The development of recombinant antibodies against C19orf47 would significantly improve reproducibility

Current research suggests that genetic validation approaches, particularly using CRISPR knockout controls, provide the most reliable method for ensuring antibody specificity and reproducibility .

How might single-cell analysis techniques benefit from improved C19orf47 antibodies?

Single-cell technologies are revolutionizing our understanding of cellular heterogeneity, and improved C19orf47 antibodies could enhance these approaches:

Applications in Single-Cell Research:

• Mass Cytometry (CyTOF):

  • Metal-conjugated C19orf47 antibodies could enable quantitative analysis at single-cell resolution

  • Integration with other protein markers to create comprehensive cellular phenotypes

  • Requires highly specific antibodies with minimal background

• Single-Cell Proteomics:

  • Antibody-based enrichment of C19orf47 and its interacting partners

  • Analysis of post-translational modifications at single-cell level

  • Correlation of C19orf47 expression with cellular states

• Spatial Transcriptomics/Proteomics:

  • Localization of C19orf47 within tissue architecture

  • Correlation with spatial gene expression patterns

  • Understanding cell-type specific expression in complex tissues

• Multiplex Imaging:

  • Integration of C19orf47 detection with multiple cellular markers

  • Analysis of subcellular localization in relation to organelles and other proteins

  • Cyclic immunofluorescence approaches for comprehensive protein localization

Technical Requirements:

• Highly specific antibodies validated in multiple systems

• Compatible with fixation and permeabilization protocols used in single-cell applications

• Minimal cross-reactivity with other cellular components

• Consistent performance across experimental conditions

Improved C19orf47 antibodies could enable researchers to better understand the protein's expression patterns and functions in heterogeneous cell populations, potentially revealing cell type-specific roles that would be masked in bulk analysis approaches.

What are the challenges and opportunities in developing therapeutic applications targeting C19orf47?

While C19orf47 is primarily a research target currently, understanding the potential therapeutic relevance presents both challenges and opportunities:

Current Challenges:

• Limited Functional Understanding:

  • C19orf47's precise biological function remains poorly characterized

  • Unclear disease associations make therapeutic targeting speculative

  • Potential off-target effects need thorough investigation

• Technical Limitations:

  • Current antibodies are research tools, not therapeutic agents

  • Intracellular location may limit accessibility for antibody therapeutics

  • Specificity across tissues requires extensive validation

• Biological Complexity:

  • Potential redundancy with related proteins

  • Unknown developmental or homeostatic roles

  • Tissue-specific functions that could impact safety profile

Future Opportunities:

• Disease Association Studies:

  • Systematic analysis of C19orf47 expression across disease states

  • Genetic association studies to identify potential links to pathologies

  • Functional studies to establish causal relationships

• Therapeutic Modality Development:

  • Antibody-based approaches:

    • Antibody-drug conjugates if C19orf47 is cell-surface accessible

    • Intrabodies for intracellular targeting

  • Non-antibody approaches:

    • Small molecule modulators of function

    • RNA-based therapeutics (siRNA, antisense oligonucleotides)

    • PROTAC-based targeted degradation

• Biomarker Applications:

  • Development of diagnostic antibodies if disease associations are established

  • Monitoring treatment responses in relevant conditions

  • Patient stratification based on C19orf47 expression or modification patterns

As research advances, improved understanding of C19orf47's biological function will better inform potential therapeutic applications. Currently, the focus remains on basic characterization using research antibodies to establish foundational knowledge.

How can researchers contribute to improving the reproducibility of C19orf47 antibody-based research?

Improving reproducibility in antibody-based research requires collective effort from the scientific community:

Best Practices for Researchers:

• Comprehensive Reporting:

  • Always include complete antibody information (manufacturer, catalog number, lot number)

  • Provide detailed validation methods specific to each application

  • Document all experimental conditions, including blocking reagents, incubation times, etc.

  • Share raw data and images when possible

• Rigorous Validation:

  • Implement genetic approaches (KO controls) whenever possible

  • Use multiple antibodies targeting different epitopes

  • Validate across all experimental applications

  • Report negative results from validation experiments

• Community Engagement:

  • Contribute validation data to public repositories

  • Report antibody performance issues to manufacturers

  • Collaborate on antibody characterization efforts

  • Participate in reproducibility initiatives

Institutional and Systemic Improvements:

• Standardization Efforts:

  • Adopt community-developed standard operating procedures

  • Implement minimum validation requirements for publication

  • Establish shared resources for antibody validation

  • Create centralized databases of validation results

• Education and Training:

  • Improve training in antibody validation techniques

  • Increase awareness of reproducibility challenges

  • Develop curricula focused on rigorous antibody usage

  • Share troubleshooting strategies and negative results

• Technological Advancements:

  • Transition to renewable antibody sources (recombinant antibodies)

  • Develop improved validation technologies

  • Create shared cell line resources for validation

  • Implement automated validation pipelines

Research has shown that genetic validation approaches provide the most reliable results, with studies indicating that antibodies validated using knockout controls perform significantly better across applications than those validated using other methods . By implementing these approaches for C19orf47 antibodies, researchers can substantially improve experimental reproducibility.

What are the best practices for selecting and validating C19orf47 antibodies for specific applications?

Based on current evidence and expert recommendations, researchers should follow these best practices:

Selection Guidelines:

Validation Hierarchy:

• Gold Standard: Testing against genetic knockout controls

• Strong Evidence: Multiple antibodies showing consistent results

• Supporting Evidence: Orthogonal validation (e.g., correlation with RNA)

• Minimum Requirement: Basic specificity controls (blocking peptide, isotype control)

Application-Specific Recommendations:

• For C19orf47 Western blot: Start with antibodies validated against knockout controls, optimize dilution (typically 1:500-1:2000), and include positive control lysates

• For C19orf47 IHC: Test multiple antibodies if possible, optimize antigen retrieval, and include tissue controls (testis and small intestine show detectable expression)

• For C19orf47 IP: Carefully optimize antibody-bead coupling, use stringent washing, and confirm enrichment relative to starting material

Studies have consistently shown that genetic validation approaches provide the most reliable antibody characterization, with significantly higher confirmation rates than orthogonal methods, particularly for immunofluorescence applications .

What are the key considerations for experimental design when working with C19orf47 antibodies?

Proper experimental design is crucial for generating reliable results with C19orf47 antibodies:

Experimental Planning:

• Control Selection:

  • Positive controls: Samples with confirmed C19orf47 expression

  • Negative controls: Knockout samples, tissues without expression, blocking peptide controls

  • Procedural controls: Primary antibody omission, isotype controls

• Sample Preparation:

  • Optimize protein extraction methods for Western blot

  • Standardize fixation procedures for IHC/IF

  • Consider tissue/cell-specific requirements

• Antibody Selection:

  • Match antibody characteristics to application requirements

  • Consider using multiple antibodies targeting different epitopes

  • Evaluate validation evidence critically

Technical Execution:

• Protocol Optimization:

  • Perform antibody titration to determine optimal concentration

  • Test different blocking reagents to minimize background

  • Optimize incubation times and temperatures

• Data Collection:

  • Collect data from multiple biological replicates

  • Include technical replicates to assess variability

  • Document all experimental parameters

• Analysis Considerations:

  • Use appropriate quantification methods

  • Apply consistent analysis criteria across samples

  • Account for potential batch effects

Interpretation Guidelines:

• Result Evaluation:

  • Consider alternative explanations for unexpected results

  • Validate findings using complementary approaches

  • Place findings in context of current knowledge

• Limitations Assessment:

  • Acknowledge technical limitations of antibody-based methods

  • Consider potential cross-reactivity issues

  • Recognize constraints in sensitivity and specificity

Research has demonstrated that antibodies validated using genetic approaches (particularly CRISPR knockouts) provide substantially more reliable results than those validated through other methods . When working with C19orf47 antibodies, this validation should be a primary consideration in experimental design.

What emerging technologies are likely to complement or enhance antibody-based research on C19orf47?

The landscape of protein research is evolving rapidly, with several emerging technologies poised to enhance our understanding of C19orf47:

Complementary Technologies:

• CRISPR-Based Approaches:

  • Endogenous tagging of C19orf47 for visualization and purification

  • CRISPRi/CRISPRa for modulating expression levels

  • Base editing for introducing specific mutations

  • Screening platforms to identify functional interactions

• Advanced Imaging Techniques:

  • Super-resolution microscopy for precise localization

  • Live-cell imaging with genetically encoded tags

  • Correlative light and electron microscopy

  • Expansion microscopy for improved spatial resolution

• Proteomics Advancements:

  • Targeted mass spectrometry for absolute quantification

  • Proximity labeling approaches (BioID, APEX) for interaction studies

  • Protein correlation profiling for complex membership

  • Single-cell proteomics for heterogeneity analysis

• Functional Genomics Integration:

  • Multi-omics approaches combining antibody data with genomics/transcriptomics

  • Pooled genetic screens to identify C19orf47-related pathways

  • Spatial transcriptomics correlated with protein localization

  • Systems biology modeling of functional networks

Future Directions for C19orf47 Research:

• Development of highly specific recombinant antibodies

• Integration of antibody-based detection with complementary technologies

• Establishment of definitive subcellular localization and interaction partners

• Characterization of potential tissue-specific functions

These emerging technologies will likely provide complementary approaches to traditional antibody-based methods, enhancing specificity, sensitivity, and biological context. By combining multiple methodologies, researchers can build more comprehensive understanding of C19orf47's biological functions and potential disease associations.