Recombinant Human Putative protein FAM106C (FAM106CP)

What is the current understanding of FAM106C within the human proteome?

FAM106C (Family with sequence similarity 106 member C, pseudogene) is classified as a putative protein with limited characterization in the scientific literature. As a pseudogene product, it belongs to a category of genomic elements historically considered non-functional but increasingly recognized for potential regulatory roles. Current research methodologies for studying putative proteins like FAM106C typically involve comparative genomics, transcriptomics analysis, and recombinant expression systems to determine if the protein is expressed in vivo and what functions it might serve. When designing studies around FAM106C, researchers should implement multiple detection methods to verify expression and avoid relying solely on computational predictions.

How should researchers approach experimental design when studying a putative protein like FAM106C?

Effective experimental design for studying putative proteins requires careful planning to maximize resource efficiency while reducing experimental bias. Key considerations include:

• Implementing blocking techniques to group similar experimental units together, reducing variability within each block and making treatment effects easier to detect

• Ensuring statistical power through appropriate sample sizing based on anticipated effect sizes

• Employing randomization strategies to minimize bias in treatment allocation

• Using blinding techniques when evaluating results to prevent observer bias

• Incorporating appropriate positive and negative controls specific to protein expression studies

A comprehensive experimental approach should combine computational prediction, mRNA expression analysis, and protein detection methods to establish whether FAM106C is genuinely expressed or if it represents a non-translated pseudogene. This multi-faceted approach helps distinguish between statistical noise and true biological signal .

What expression systems are most suitable for producing recombinant FAM106C protein?

The selection of an expression system for recombinant FAM106C production should be informed by protein characteristics and research objectives:

Similar to strategies employed for aminoacyl-tRNA synthetases, selected domains of FAM106C can be produced recombinantly in E. coli for use as antigens in antibody production or for initial characterization . For full-length protein with proper folding and modifications, mammalian expression systems are recommended, particularly for functional studies.

What purification strategies are recommended for recombinant FAM106C?

Purification of recombinant FAM106C typically involves a multi-step approach:

• Initial Capture: Affinity chromatography using a fusion tag (His, GST, or MBP) enables efficient capture from crude lysate

• Intermediate Purification: Ion exchange chromatography based on predicted isoelectric point

• Polishing: Size exclusion chromatography to achieve high purity and remove aggregates

The purification protocol should include validation steps at each stage, including SDS-PAGE, Western blotting, and mass spectrometry to confirm identity. When designing purification strategies, researchers should consider potential structural characteristics that might influence protein behavior during purification, such as hydrophobic regions or intrinsically disordered domains.

How can researchers effectively validate antibodies against FAM106C?

Antibody validation is critical for studies of putative proteins like FAM106C to ensure specificity and reproducibility. A comprehensive validation approach should include:

• Cross-reactivity testing: Screen against related family members to establish specificity

• Immunoprecipitation followed by mass spectrometry (IP-MS): Verify capture of endogenous protein from mammalian cell lysates

• Immunofluorescence assays: Confirm subcellular localization patterns

• Knockout/knockdown controls: Test antibody specificity using CRISPR/Cas9-edited cell lines lacking FAM106C expression

• Epitope mapping: Identify the specific region recognized by the antibody

The validation approach used by researchers studying aminoacyl-tRNA synthetases provides an excellent model, where antibodies were systematically tested through binding assays against purified antigen followed by IP-MS to capture endogenous protein from mammalian cell lysates .

What approaches should be used to investigate potential post-translational modifications of FAM106C?

Investigation of post-translational modifications (PTMs) in FAM106C requires a systematic approach:

• Computational prediction: Utilize algorithms to identify potential modification sites

• Mass spectrometry analysis: Employ high-resolution MS/MS to identify and map modifications

• Site-directed mutagenesis: Mutate predicted modification sites to assess functional impact

• Specific PTM antibodies: Use antibodies that recognize common modifications (phosphorylation, ubiquitination, etc.)

For phosphorylation specifically, researchers should consider both conventional kinases and secretory pathway kinases like Fam20C, which has been shown to phosphorylate numerous secreted proteins at S-x-E/pS motifs . If FAM106C contains such motifs, it could be a substrate for this kinase family.

What experimental approaches are most effective for determining FAM106C protein-protein interactions?

Protein interaction studies for FAM106C should employ multiple complementary methods:

• Co-immunoprecipitation (Co-IP): Using validated antibodies to capture endogenous FAM106C and associated proteins

• Proximity labeling: BioID or APEX2 fusion proteins to identify neighboring proteins in living cells

• Yeast two-hybrid screening: Systematic identification of potential binding partners

• Pull-down assays: Using recombinant FAM106C as bait

• Mass spectrometry analysis: Identification of co-precipitated proteins

When analyzing interaction results, researchers should be cautious about false positives and implement appropriate controls. The approach used for multi-tRNA synthetase complex members, where antibodies targeting individual components successfully co-immunoprecipitated the entire complex, demonstrates how interaction studies can reveal functional complexes .

How should researchers approach the analysis of FAM106C expression data across different tissues?

Analysis of FAM106C expression requires careful attention to data integrity and statistical methods:

• Data normalization: Account for technical variations between samples

• Statistical testing: Apply appropriate tests based on data distribution

• Multiple testing correction: Implement FDR or Bonferroni correction when comparing across multiple tissues

• Validation: Confirm RNA-seq or microarray findings with qPCR, Western blot, or immunohistochemistry

• Single-cell analysis: Consider cellular heterogeneity within tissues

Researchers should be aware that the environment or context in which data was collected could influence analysis results . For example, different RNA extraction methods might yield varying results, and researchers should account for these methodological differences when comparing across studies.

How can researchers distinguish between true biological effects and statistical artifacts when studying FAM106C?

Distinguishing signal from noise in FAM106C studies requires rigorous analytical approaches:

• Adequate sample size: Ensure sufficient statistical power to detect true effects

• Appropriate controls: Include positive and negative controls in all experiments

• Effect size consideration: Focus on biological significance, not just statistical significance

• Replication: Validate findings across independent experiments and different methodologies

• Transparent reporting: Document all data preprocessing steps and exclusion criteria

What strategies help resolve contradictory findings about FAM106C in the literature?

When confronted with contradictory research findings:

• Methodological assessment: Evaluate differences in experimental approaches

• Cell/tissue specificity: Consider whether contradictions might reflect genuine biological differences across systems

• Temporal dynamics: Assess whether contradictions relate to different time points or developmental stages

• Literature meta-analysis: Systematically compare methodologies and findings across studies

• Collaborative verification: Engage with other researchers to replicate key experiments

For putative proteins like FAM106C, contradictions often arise from differences in detection methods, expression systems, or antibody specificity. Researchers should maintain a sufficient and accurate paper trail of how data was manipulated for future review, as recommended by Shamoo (1989) .

What quality control measures are essential when working with recombinant FAM106C?

Rigorous quality control ensures reliable research outcomes:

The integrity of recombinant FAM106C should be verified before use in downstream applications. For proteins expressed in E. coli, endotoxin removal is particularly important for cell-based assays to prevent non-specific effects.

How can researchers troubleshoot low expression or insolubility of recombinant FAM106C?

When encountering expression challenges:

• Codon optimization: Adapt codons to expression host preference

• Fusion partners: Test solubility-enhancing tags (MBP, SUMO, Thioredoxin)

• Expression conditions: Optimize temperature, induction timing, and media composition

• Construct design: Express individual domains or truncated versions

• Host strain selection: Test multiple strains with different folding capabilities

For proteins that remain challenging, consider cell-free expression systems or inclusion body refolding protocols. The approach used for producing domains of aminoacyl-tRNA synthetases in E. coli could serve as a model, where researchers successfully expressed difficult proteins for antibody generation .

What considerations are important when designing CRISPR/Cas9 genome editing experiments targeting FAM106C?

CRISPR/Cas9 experiments targeting FAM106C require careful design:

• Guide RNA design: Select targets with minimal off-target potential

• Knockout verification: Implement genomic PCR, RT-PCR, and Western blotting to confirm editing

• Phenotypic characterization: Use multiple assays to identify subtle phenotypes

• Rescue experiments: Reintroduce FAM106C to confirm phenotype specificity

• Control cell lines: Generate and characterize multiple clonal lines

CRISPR/Cas9 genome editing has been successfully employed to study protein function, as demonstrated in research on Fam20C where this approach helped identify more than 100 secreted phosphoproteins as genuine substrates .

What methodological approaches are recommended for studying the potential role of FAM106C in disease processes?

Disease-focused research on FAM106C should employ:

• Patient sample analysis: Compare expression levels in healthy vs. diseased tissues

• Genetic association studies: Analyze correlations between FAM106C variants and disease outcomes

• Functional assays: Develop disease-relevant cellular assays to assess impact

• Animal models: Generate knockout or transgenic models if justified by preliminary data

• Pathway analysis: Examine relationships with known disease mechanisms

Like aminoacyl-tRNA synthetases, which were long viewed as mere housekeeping proteins but later linked to autoimmune diseases, cancer, and neurological disorders , putative proteins like FAM106C might have unrecognized roles in disease processes that can only be uncovered through systematic investigation.