FAM71C Antibody

What is FAM71C and what applications is the FAM71C antibody suitable for?

FAM71C (Family with sequence similarity 71 member C) is a human protein encoded by the FAM71C gene (GeneID: 196472). The FAM71C antibody is primarily used for detecting this protein in research contexts. Based on validated testing, this antibody is suitable for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) applications . These techniques allow researchers to detect and quantify the presence of FAM71C in various sample types. When planning experiments, it's important to consider that while these applications have been validated, optimization may be required for specific experimental conditions and sample types.

What are the key specifications of commercially available FAM71C antibodies?

The commercially available FAM71C antibody typically has the following specifications:

These specifications are crucial for determining the antibody's suitability for specific experimental designs . Researchers should carefully consider these parameters when designing protocols to ensure optimal performance.

How should FAM71C antibody be stored and handled to maintain its efficacy?

Proper storage and handling of FAM71C antibody is critical for maintaining its functionality and specificity. The antibody should be aliquoted upon receipt and stored at -20°C to prevent repeated freeze/thaw cycles, which can significantly degrade antibody performance . When preparing working solutions, use sterile techniques and appropriate buffer conditions. The shipping buffer (0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol) provides stability, but researchers may need to adjust buffer conditions for specific applications. Maintaining proper storage conditions is essential, as antibody degradation can lead to increased background signal, reduced sensitivity, and potentially misleading experimental results.

What controls should be included when using FAM71C antibody in Western blot experiments?

When designing Western blot experiments with FAM71C antibody, including appropriate controls is essential for result validation:

• Positive control: Include samples known to express FAM71C (based on UniProt data, Q8NEG0). Human tissue or cell lines with verified FAM71C expression should be used.

• Negative control: Include samples known not to express FAM71C or samples from FAM71C knockout models.

• Loading control: Use antibodies against housekeeping proteins (e.g., β-actin, GAPDH) to normalize for protein loading variations.

• Isotype control: Include a non-specific IgG from the same host species (rabbit) at the same concentration to assess non-specific binding.

• Blocking peptide control: If available, pre-incubate FAM71C antibody with the immunizing peptide before application to verify specificity.

For Western blots specifically, optimization of antibody dilution within the recommended range (1/500 - 1/2000) is necessary , as is determination of appropriate blocking conditions to minimize background. This systematic approach helps distinguish genuine FAM71C detection from artifacts or non-specific signals.

How can researchers optimize immunofluorescence protocols using FAM71C antibody?

While immunofluorescence is not listed among the validated applications for this FAM71C antibody, researchers might still want to explore this application. Drawing from established immunofluorescence methodologies as described in the literature , the following optimization steps are recommended:

• Fixation optimization: Test multiple fixation methods (4% paraformaldehyde, methanol, or acetone) to determine which best preserves FAM71C epitopes.

• Permeabilization testing: Compare different permeabilization agents (0.1% Triton X-100, 0.1% Saponin) and durations to optimize antibody access to intracellular FAM71C.

• Blocking optimization: Test various blocking solutions (5% BSA, 5-10% normal serum from the secondary antibody host species) to reduce background.

• Antibody dilution series: Perform a titration series starting at 1:100 and extending beyond the recommended WB dilutions (1:500-1:2000) .

• Signal amplification: Consider secondary antibody selection carefully, and potentially employ signal amplification methods if FAM71C expression is low.

Include positive control samples where FAM71C expression is well-established and negative controls to assess background and non-specific binding. Counterstain nuclei with DAPI and include appropriate cytoskeletal markers to provide cellular context for FAM71C localization.

How can FAM71C antibody be utilized in proteomics research and what special considerations apply?

FAM71C antibody can be valuable in proteomics research, particularly in studies employing techniques like immunoprecipitation followed by mass spectrometry (IP-MS). When incorporating FAM71C antibody into proteomics workflows:

• Antibody-based enrichment: FAM71C antibody can be used for immunoprecipitation to enrich FAM71C and its binding partners. Consider crosslinking the antibody to beads (e.g., Protein G magnetic beads) to prevent antibody contamination in the eluted sample .

• Validation in LC-MS/MS contexts: If using FAM71C antibody in conjunction with mass spectrometry, validate the specificity of enrichment using Western blotting before proceeding to MS analysis. This is particularly important given that proteomics techniques like iTRAQ combined with LC-MS/MS are sensitive to non-specific binding .

• Interaction network analysis: After IP-MS, analyze potential FAM71C interactors using bioinformatics tools to construct interaction networks. Consider the subcellular localization and known functions of identified partners to build biologically relevant models.

• Quantitative proteomics: For differential expression studies, techniques like iTRAQ labeling can be used to compare FAM71C levels or FAM71C-associated proteins across different experimental conditions .

The interpretation of proteomics data requires careful consideration of potential false positives arising from non-specific binding. Therefore, stringent washing procedures and appropriate negative controls are essential for generating reliable results.

What approaches are recommended for troubleshooting non-specific binding or weak signals when using FAM71C antibody?

When encountering issues with FAM71C antibody performance, a systematic troubleshooting approach is essential:

• For non-specific binding issues:

  • Increase blocking time or concentration (try 5% BSA or 5% milk in TBS-T)

  • Increase washing stringency (more washes, higher salt concentration)

  • Optimize antibody dilution (test dilutions across and beyond the recommended 1:500-1:2000 range)

  • Pre-absorb the antibody with cell/tissue lysate lacking FAM71C

  • Use gradient SDS-PAGE to better separate proteins of similar molecular weight

• For weak signal issues:

  • Increase protein loading amount (up to 50-100 μg per lane for Western blot)

  • Reduce antibody dilution (start at 1:250 if the recommended 1:500 gives weak signals)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use more sensitive detection methods (chemiluminescent substrate with extended exposure)

  • Ensure sample proteins are properly denatured and reduced

  • Consider enrichment strategies for low-abundance proteins

• General optimization:

  • Verify target protein expression in your sample type

  • Test different lysis buffers to optimize protein extraction

  • Consider the effect of post-translational modifications on epitope accessibility

  • Verify antibody quality (avoid repeated freeze-thaw cycles)

Document all troubleshooting steps methodically to establish an optimized protocol for your specific experimental system.

How can FAM71C antibody be adapted for use in high-throughput screening or array-based technologies?

Adapting FAM71C antibody for high-throughput or array-based applications requires careful optimization:

• Antibody microarrays: When spotting FAM71C antibody onto array surfaces, maintain consistent antibody concentration and spotting conditions. Optimize blocking and washing steps to minimize background while preserving specific signals.

• Reverse-phase protein arrays (RPPA): For RPPA, where cell/tissue lysates are spotted and probed with antibodies including FAM71C, validate antibody specificity thoroughly with Western blot before RPPA application. Test multiple antibody dilutions to establish the linear detection range.

• High-content screening: When using FAM71C antibody in automated microscopy workflows, establish standardized protocols for fixation, permeabilization, and antibody incubation that can be consistently applied across plates. Develop robust image analysis parameters to quantify FAM71C signals.

• Adaptation to phage display systems: Drawing from scFv library screening methodologies , consider developing or selecting FAM71C-specific single-chain variable fragments that may offer advantages in certain high-throughput applications.

• Multiplexed detection: When combining FAM71C detection with other targets, carefully select antibodies raised in different host species to avoid cross-reactivity of secondary antibodies. Alternatively, use directly conjugated primary antibodies.

For all high-throughput adaptations, substantial initial validation is required to establish the specificity and sensitivity of the assay under the modified conditions, followed by statistical validation of the reproducibility and robustness of the method.

What considerations should researchers take into account when analyzing FAM71C expression in different cell types or tissues?

When analyzing FAM71C expression across diverse biological samples:

• Baseline expression profiling: Before comparative studies, establish baseline FAM71C expression levels in your biological system. Consult UniProt (Q8NEG0) and public gene expression databases for tissue distribution information.

• Normalization strategies: Carefully select appropriate housekeeping genes or proteins for normalization, as their expression may vary across tissues or experimental conditions. Consider using multiple reference genes/proteins and geometric averaging for more robust normalization.

• Isoform awareness: Be cognizant that FAM71C may have multiple isoforms in different tissues. The antibody, raised against amino acids 1-241 , may detect specific isoforms differentially. Western blot analysis should account for potential molecular weight variations.

• Post-translational modifications: Consider how tissue-specific post-translational modifications might affect epitope recognition. Phosphorylation, glycosylation, or other modifications may alter antibody binding efficiency.

• Sample preparation optimization: Different tissues may require specific lysis or extraction procedures to effectively solubilize FAM71C. Optimize protein extraction protocols for each tissue type to ensure comparable recovery.

• Cross-reactivity assessment: While the antibody is reported to be human-specific , evaluate potential cross-reactivity if working with samples from other species, particularly in evolutionary studies.

A multi-method approach combining protein detection (Western blot, ELISA) with mRNA analysis (qRT-PCR) provides the most comprehensive understanding of FAM71C expression patterns.

How should researchers interpret conflicting results when comparing FAM71C detection across different experimental methods?

When faced with discrepancies in FAM71C detection across methods:

• Methodological limitations assessment: Each detection method has inherent strengths and limitations. Western blotting provides information about molecular weight but may be affected by protein denaturation; ELISA offers quantitative analysis but may detect degraded proteins; immunofluorescence provides spatial information but may suffer from fixation artifacts.

• Epitope accessibility considerations: The polyclonal antibody recognizes epitopes within the recombinant human FAM71C protein (1-241AA) . Different experimental conditions may affect epitope accessibility—native conditions may conceal epitopes that are exposed after denaturation.

• Systematic validation approach: When results conflict, implement a systematic validation strategy:

  • Verify antibody specificity using positive and negative controls

  • Test multiple antibody concentrations

  • Employ alternative detection methods (fluorescent vs. chromogenic)

  • Consider using a second antibody targeting a different FAM71C epitope

  • Complement protein studies with mRNA analysis (qRT-PCR)

• Statistical analysis: Apply appropriate statistical tests to determine if observed differences are significant. For multiple comparisons, use methods that control for family-wise error rate or false discovery rate.

• Literature reconciliation: Contextualize your findings within the published literature, recognizing that biological variability, technical differences, and cellular contexts can all contribute to apparently conflicting results.

What bioinformatic approaches can enhance the integration of FAM71C antibody-derived data with other -omics datasets?

Integrating FAM71C antibody data with other -omics information requires sophisticated bioinformatic strategies:

• Multi-omics data integration: Combine FAM71C protein expression data with:

  • Transcriptomic data to assess correlation between mRNA and protein levels

  • Phosphoproteomic data to identify post-translational modifications

  • Interactomics data to place FAM71C in protein-protein interaction networks

  • Functional genomics data (e.g., CRISPR screens) to understand functional relationships

• Network analysis techniques: Apply methods like weighted gene co-expression network analysis (WGCNA) to identify modules of genes/proteins whose expression patterns correlate with FAM71C across conditions or tissues.

• Pathway enrichment analysis: Use databases like KEGG, Reactome, or Gene Ontology to identify biological processes enriched among genes/proteins correlated with FAM71C expression. This can provide functional context, similar to approaches used in proteomics studies .

• Visualization strategies: Employ advanced visualization tools (e.g., Cytoscape, R packages like ggplot2, heatmaps) to represent complex relationships between FAM71C and other molecular features.

• Machine learning approaches: Consider supervised or unsupervised machine learning methods to:

  • Identify patterns in complex multi-omics datasets

  • Predict functional relationships

  • Classify samples based on FAM71C and related molecular features

• Single-cell analysis integration: If working with single-cell data, correlate FAM71C protein levels with cell-type-specific markers to understand expression patterns at single-cell resolution.

These bioinformatic approaches can transform isolated FAM71C antibody data into insights within broader biological systems and networks.

How might FAM71C antibody be utilized in studies of disease mechanisms or biomarker development?

FAM71C antibody has potential applications in disease research and biomarker development:

• Expression profiling in disease states: Compare FAM71C expression between normal and diseased tissues to identify potential associations with pathological processes. Use standardized antibody-based assays like immunohistochemistry on tissue microarrays or quantitative Western blotting.

• Biomarker validation methodology: If preliminary data suggests FAM71C as a potential biomarker:

  • Develop standardized ELISA protocols using the FAM71C antibody

  • Establish reference ranges in healthy populations

  • Assess sensitivity and specificity in disease detection

  • Evaluate reproducibility across multiple laboratories

  • Compare performance against existing biomarkers

• Mechanistic studies: Use FAM71C antibody to:

  • Monitor changes in FAM71C expression during disease progression

  • Identify alterations in subcellular localization in disease states

  • Detect post-translational modifications specific to pathological conditions

  • Identify disease-specific interaction partners through co-immunoprecipitation

• Therapeutic target assessment: If FAM71C emerges as a potential therapeutic target, use the antibody to:

  • Validate target engagement in drug screens

  • Monitor changes in expression or modification state following treatment

  • Develop companion diagnostics for targeted therapies

While specific disease associations for FAM71C are not well-established in the provided literature, systematic approaches using validated antibody-based methods can uncover potential roles in human disease.

What considerations should be taken into account when adapting FAM71C antibody for use in single-cell protein analysis techniques?

Adapting FAM71C antibody for single-cell protein analysis requires careful optimization:

• Mass cytometry (CyTOF) considerations:

  • Metal conjugation must preserve epitope recognition

  • Thorough titration is essential to determine optimal antibody concentration

  • Include appropriate isotype controls conjugated to the same metal

  • Validate specificity in positive and negative control cell populations

• Single-cell Western blot adaptation:

  • Optimize lysis conditions to maximize protein release while maintaining separation between cells

  • Adjust antibody concentration for the reduced protein amount per cell

  • Extend incubation times to enhance sensitivity

  • Use highly sensitive detection methods (e.g., Tyramide Signal Amplification)

• Microfluidic antibody capture techniques:

  • Determine optimal surface chemistry for antibody immobilization

  • Optimize flow rates and capture times

  • Develop appropriate washing protocols to reduce background without losing sensitivity

  • Calibrate detection systems using recombinant FAM71C standards

• Imaging-based single-cell proteomics:

  • Adapt immunofluorescence protocols to maintain cellular morphology

  • Optimize fixation and permeabilization for FAM71C epitope preservation

  • Consider cyclic immunofluorescence or multiplexed ion beam imaging for co-detection with other proteins

  • Implement robust image analysis algorithms for accurate quantification

• Quality control metrics:

  • Develop appropriate positive and negative controls for each single-cell technique

  • Implement batch correction methods for large-scale studies

  • Establish thresholds for distinguishing positive from negative cells

These advanced applications require rigorous validation and often necessitate higher antibody concentrations than traditional bulk methods to achieve adequate sensitivity at the single-cell level.

What are the most critical factors for researchers to consider when selecting and validating FAM71C antibody for their specific applications?

When selecting and validating FAM71C antibody for research:

• Application compatibility: Ensure the antibody has been validated for your intended application. The FAM71C antibody discussed here is validated for ELISA and Western blot applications , and adaptation to other techniques will require thorough validation.

• Epitope considerations: The antibody is raised against recombinant human FAM71C protein (1-241AA) , so experiments requiring recognition of specific domains should verify that the relevant epitopes are included in this region.

• Species reactivity: This antibody is specifically reactive with human FAM71C . Research involving other species would require verification of cross-reactivity or selection of species-specific alternatives.

• Validation strategy: Implement a multi-step validation:

  • Positive and negative controls to confirm specificity

  • Concentration optimization for your specific application

  • Reproducibility testing across multiple experiments

  • Comparison with alternative detection methods when possible

• Technical expertise requirements: Consider the technical complexity of your chosen application and ensure appropriate expertise and equipment are available.

• Experimental context: The biological context of your research question should inform antibody selection—consider expression levels, potential interfering factors, and the cellular or tissue environment in which you're studying FAM71C.

How can researchers effectively document and report FAM71C antibody-based methods to ensure reproducibility in the scientific literature?

To ensure reproducibility in FAM71C antibody research:

• Comprehensive antibody documentation: Report:

  • Complete antibody identifier information (manufacturer, catalog number, lot number)

  • Host species, clonality, and immunogen details (recombinant human FAM71C protein 1-241AA)

  • RRID (Research Resource Identifier) if available

  • Validation data supporting use in the reported application

• Detailed methodology reporting:

  • Precise antibody dilutions used (not just manufacturer's recommended range of 1:500-1:2000)

  • Complete buffer compositions (beyond simply "PBS" or "blocking buffer")

  • Incubation times and temperatures

  • Sample preparation procedures, including fixation and permeabilization methods

  • Image acquisition parameters for microscopy-based methods

  • Data processing workflows, including normalization methods

• Controls documentation:

  • Describe all positive and negative controls

  • Include representative images of controls in publications

  • Document how specificity was verified

• Quantification transparency:

  • Clearly explain quantification methods

  • Report biological and technical replicates

  • Provide raw data when possible or upon request

  • Document statistical approaches, including handling of outliers

• Limitation acknowledgment:

  • Discuss potential limitations of the antibody-based methods

  • Address alternative interpretations of results

  • Suggest complementary approaches for validation