Recombinant Human Protein FAM194A (FAM194A)

What is the basic structure and function of human FAM194A protein?

Human FAM194A belongs to the family with sequence similarity 194 proteins. Based on comparative analysis with other FAM proteins, it likely contains specific functional domains that may be involved in cellular signaling or regulatory processes. While direct structural data for human FAM194A is limited, sequence analysis suggests several potential domains that could mediate protein-protein interactions.

For researchers beginning work with this protein, it's recommended to perform bioinformatic analyses to predict functional domains and potential post-translational modification sites, which can guide experimental design. The protein may share functional characteristics with other members of the FAM protein superfamily, which generally have roles in diverse cellular processes including development, metabolism, and immune response.

What expression systems are most effective for producing recombinant human FAM194A?

For optimal expression of functional human FAM194A protein, mammalian expression systems like HEK293 cells are generally preferred over bacterial systems. This preference is based on the need for proper post-translational modifications and protein folding.

Methodologically, researchers should consider:

• Using codon-optimized sequences for expression in the chosen host system

• Including purification tags (His, Fc, or Avi-tags) as demonstrated in mouse FAM194A expression

• Employing mammalian expression vectors with strong promoters (e.g., CMV)

• Testing different cell lines if initial expression attempts yield low protein levels

Based on successful approaches with related proteins and mouse FAM194A, stable transfection of HEK293 cells has shown good results for producing recombinant proteins with proper folding and post-translational modifications .

How can I validate the specificity and functionality of custom antibodies against human FAM194A?

Validating antibody specificity for FAM194A requires multiple complementary approaches:

• Overexpression validation: Express FAM194A-EGFP fusion protein in cells that do not endogenously express FAM194A, then confirm antibody detection of the fusion protein by western blot and immunofluorescence

• Knockout validation: Include samples from FAM194A knockout systems as negative controls to confirm absence of signals

• Cross-reactivity testing: Test antibodies against related FAM family proteins to ensure specificity

• Multiple antibody concordance: Use antibodies targeting different epitopes of FAM194A and verify consistent staining patterns

Researchers should acknowledge potential limitations in antibody sensitivity, as seen with related proteins where detection in western blot may require overexpression systems . When publishing, include detailed validation methods and acknowledge any limitations in antibody performance.

What are the optimal methods for studying protein-protein interactions involving human FAM194A?

Based on successful approaches with related FAM proteins, a multi-method strategy is recommended:

• Proximity-dependent biotin identification (BioID): This technique allows for identification of proteins that transiently interact with or are in close proximity to FAM194A. The approach involves:

  • Creating a fusion protein with FAM194A and a promiscuous biotin ligase

  • Expressing this construct in relevant cell types

  • Analyzing biotinylated proteins by mass spectrometry

• Co-immunoprecipitation followed by mass spectrometry: This classical approach complements BioID and can validate key interactions:

  • Use specific antibodies against FAM194A or epitope tags

  • Perform sufficient washing steps to reduce non-specific binding

  • Confirm interactions by reciprocal co-immunoprecipitation

• Validation approaches:

  • Confirm key interactions using microscopy-based co-localization studies

  • Use gene correlation analysis from single-cell transcriptomics data to support protein interaction findings

  • Perform functional validation through genetic manipulation of interaction partners

STRING analysis of overlapping hits from both BioID and immunoprecipitation can reveal functional networks, as demonstrated with related FAM proteins that showed associations with catalytic complexes, protein transport, and specific subcellular compartments .

What techniques are most effective for studying the cellular localization of FAM194A?

A comprehensive approach to determining FAM194A localization should include:

• Immunohistochemistry and immunofluorescence:

  • Use validated antibodies against endogenous FAM194A

  • Include appropriate knockout controls to confirm specificity

  • Employ co-staining with subcellular markers to identify precise localization

• Subcellular fractionation:

  • Separate cellular components (nucleus, cytoplasm, mitochondria, etc.)

  • Perform western blot analysis of fractions to detect FAM194A

  • Include fraction-specific markers to verify separation quality

• Live-cell imaging with fluorescent fusion proteins:

  • Create N- and C-terminal fluorescent protein fusions with FAM194A

  • Compare localization patterns to ensure tag position doesn't disrupt targeting

  • Consider using photoactivatable or photoconvertible fluorescent proteins for dynamic studies

When analyzing localization, researchers should investigate potential post-translational modifications that might affect localization. For instance, analysis of related FAM proteins revealed N-myristoylation sites and phosphorylation sites that correlated with specific subcellular targeting .

What are the recommended methods for detecting FAM194A in various tissue types?

Detection of FAM194A across different tissues requires a tailored approach:

• Tissue preparation considerations:

  • For fixed tissues: Optimize fixation conditions (formalin, paraformaldehyde) and antigen retrieval methods

  • For frozen tissues: Determine optimal section thickness and fixation protocols

  • Process matched tissues from knockout animals as negative controls

• Detection methods:

  • RT-qPCR: Design specific primers that distinguish FAM194A from other FAM family members

  • RNAscope: For sensitive in situ detection of mRNA in tissues with low expression

  • Immunohistochemistry: Use DAB or fluorescence-based detection with validated antibodies

  • Western blot: May require tissue-specific protein extraction protocols

• Sensitivity enhancement strategies:

  • Consider signal amplification methods for tissues with low expression

  • Use pre-coupled magnetic beads for protein enrichment prior to detection

  • Implement proximity ligation assays for improved sensitivity and specificity

Researchers should note that detection sensitivity varies across tissues, and sensitivity limitations may necessitate the use of overexpression systems for certain applications, as observed with related FAM proteins .

How can I effectively design knockout experiments to study FAM194A function?

Based on approaches used with related FAM proteins, a comprehensive knockout study should include:

• Multiple knockout strategies:

  • CRISPR-Cas9 targeting with multiple guide RNAs to ensure complete protein loss

  • Consider conditional knockout systems for studying developmental roles

  • Include rescue experiments with wild-type protein to confirm phenotype specificity

• Phenotypic analysis considerations:

  • Monitor both basic parameters (viability, growth) and specialized functions

  • For metabolism studies: measure body weight, energy expenditure, and metabolic parameters

  • Assess tissue-specific effects, particularly in tissues with high expression

• Validation requirements:

  • Confirm knockout at both mRNA (RT-qPCR) and protein levels (western blot, immunohistochemistry)

  • Include littermate controls to minimize genetic background effects

  • Consider compensatory mechanisms by other FAM family members

When designing knockout experiments, researchers should anticipate potential metabolic phenotypes based on findings from related FAM protein knockouts, which demonstrated altered body weight and decreased energy expenditure .

What cell-based assays are most informative for studying FAM194A function?

Based on functional associations of related FAM proteins, consider these assay systems:

• Metabolic function assays:

  • Seahorse XF analysis for mitochondrial respiration and glycolytic function

  • Cellular bioenergetics measurements (ATP production, NAD+/NADH ratios)

  • Lipid metabolism assays (lipid droplet formation, fatty acid oxidation)

• Protein transport and trafficking:

  • Live-cell imaging of vesicular trafficking

  • Endocytosis and exocytosis rate measurements

  • Secretory pathway function assessment

• Cell signaling studies:

  • Phosphoproteomic analysis to identify downstream effectors

  • Luciferase reporter assays for relevant signaling pathways

  • Calcium signaling measurements if membrane or ER localization is detected

• Cell-cell interaction studies:

  • Co-culture systems with immune cells if immune-related functions are suspected

  • Assessment of paracrine signaling effects

When interpreting results, compare with STRING analysis data from related FAM proteins that revealed associations with catalytic complexes, intracellular protein transport, mitochondrial inner membrane, respiratory electron transport, and protein export .

What approaches are recommended for investigating potential roles of FAM194A in specific disease contexts?

A systematic approach to investigating disease associations should include:

• Transcriptomic and proteomic analyses:

  • Compare FAM194A expression levels across normal and disease tissues

  • Correlate expression with disease progression and patient outcomes

  • Identify co-expressed genes that may reveal functional networks

• Genetic association studies:

  • Analyze available GWAS data for SNPs in or near FAM194A

  • Perform targeted sequencing in patient cohorts with suspected associations

  • Evaluate copy number variations affecting the FAM194A locus

• Functional disease models:

  • Develop cell-based disease models with FAM194A manipulation

  • Create animal models mimicking human disease with altered FAM194A expression

  • Test pharmacological agents that might modulate FAM194A function

Given the associations of other FAM proteins with immune response and cancer progression, researchers should consider investigating FAM194A in these contexts. For example, FAM111A has demonstrated roles in tumor microenvironment and immune response in lower-grade glioma , suggesting that FAM proteins may have diverse roles in disease contexts.

What bioinformatic approaches can reveal evolutionary conservation and functional predictions for human FAM194A?

Advanced computational analysis of FAM194A should include:

• Comparative genomics:

  • Perform phylogenetic analysis across species to identify conserved domains

  • Create habitat-specific matrices to correlate protein features with environmental adaptations

  • Analyze sequence conservation patterns that might reveal functional constraints

• Structural prediction and analysis:

  • Apply AlphaFold or similar tools to predict protein structure

  • Identify potential binding pockets and interaction surfaces

  • Map conserved regions onto predicted structures to identify functional domains

• Integrative multi-omics analysis:

  • Correlate FAM194A expression with tissue-specific transcriptomes

  • Integrate proteomic interaction data with transcriptomic co-expression networks

  • Analyze epigenetic regulation of FAM194A expression across tissues and conditions

• Domain function prediction:

  • Identify domains of unknown function (e.g., DUF4619 in related proteins)

  • Perform in silico docking studies with potential interaction partners

  • Map post-translational modification sites and predict their functional impact

Researchers should note that similar analyses of related FAM proteins have revealed potential N-myristoylation sites and phosphorylation sites that correlated with specific functional adaptations and habitat-specific evolutionary patterns .

How can I investigate potential roles of FAM194A in immune response and inflammation?

Given the immune-related functions observed in other FAM family members, researchers studying FAM194A should consider:

• Immune cell-specific expression analysis:

  • Analyze FAM194A expression across immune cell subsets using single-cell RNA-seq data

  • Determine if expression changes during immune cell activation or differentiation

  • Compare expression patterns with known immune modulators

• Functional immune assays:

  • Assess effects of FAM194A overexpression or knockout on:

    • Cytokine production and secretion

    • Immune cell migration and chemotaxis

    • Antigen presentation and processing

    • Phagocytosis and efferocytosis

• In vivo immune challenge models:

  • Challenge FAM194A knockout mice with pathogens or inflammatory stimuli

  • Analyze changes in immune cell populations and inflammatory markers

  • Assess tissue-specific immune responses

When designing these studies, consider that other FAM family members (e.g., FAM111A) have shown associations with inflammatory response, immune cell populations (particularly monocytic lineage, myeloid dendritic cells), and M2 macrophage cells .

What experimental designs are most appropriate for studying potential interactions between FAM194A and other FAM family proteins?

To investigate functional relationships between FAM194A and other FAM proteins:

• Co-expression and co-localization studies:

  • Perform systematic co-expression analysis across tissues and conditions

  • Use fluorescently tagged proteins to assess subcellular co-localization

  • Apply super-resolution microscopy for detailed spatial relationship analysis

• Functional redundancy assessment:

  • Create single and combinatorial knockouts of multiple FAM family members

  • Perform rescue experiments with different family members

  • Assess compensation mechanisms through transcriptomic analysis after knockouts

• Protein-protein interaction analysis:

  • Test direct interactions using proximity ligation assays

  • Perform co-immunoprecipitation studies with multiple FAM proteins

  • Use FRET or BiFC to assess protein proximity in living cells

• Regulatory relationship investigation:

  • Analyze effects of one FAM protein on the expression of others

  • Identify shared transcriptional regulators or regulatory elements

  • Investigate coordinate regulation during development or stress responses

When designing these studies, researchers should consider that FAM proteins may have evolved specialized functions while maintaining some degree of functional overlap, potentially serving as a backup system in cellular processes.

How can FAM194A be studied in the context of cancer biomarker development?

Based on the application of other FAM proteins as cancer biomarkers, investigations of FAM194A should consider:

• Expression analysis in cancer datasets:

  • Analyze FAM194A expression across cancer types and stages

  • Correlate expression with patient survival and treatment response

  • Determine association with specific molecular subtypes of cancers

• Epigenetic regulation assessment:

  • Analyze promoter methylation status of FAM194A in different cancers

  • Determine if FAM194A could serve as a methylation marker similar to FAM19A4

  • Develop quantitative PCR assays for methylation analysis

• Functional validation in cancer models:

  • Perform knockdown or overexpression studies in cancer cell lines

  • Assess effects on proliferation, migration, invasion, and apoptosis

  • Determine impact on treatment sensitivity and resistance mechanisms

When developing such biomarkers, researchers should follow methodological approaches similar to those used for FAM19A4/miR124-2 methylation testing, which demonstrated high sensitivity for cancer detection and prognostic value .

What methodological considerations are important when studying post-translational modifications of FAM194A?

A comprehensive approach to studying FAM194A post-translational modifications includes:

• Modification site identification:

  • Use mass spectrometry-based proteomics to map modification sites

  • Compare with predicted sites from bioinformatic analysis

  • Create site-specific antibodies for key modifications

• Functional impact assessment:

  • Generate site-specific mutants (e.g., phosphomimetic or phospho-deficient)

  • Assess effects on protein localization, stability, and interactions

  • Determine impact on cellular functions and signaling pathways

• Regulatory enzyme identification:

  • Use proximity labeling methods to identify potential kinases, phosphatases, or other modifying enzymes

  • Perform in vitro modification assays to confirm direct enzyme-substrate relationships

  • Use specific inhibitors to validate relationships in cellular systems

Researchers should consider potential N-myristoylation sites and phosphorylation sites, as these have been identified in related FAM proteins and correlated with functional adaptations .

How can single-cell analysis approaches advance our understanding of FAM194A function?

Single-cell methodologies offer unique insights into FAM194A biology:

• Single-cell transcriptomics applications:

  • Analyze cell type-specific expression patterns across tissues

  • Identify co-expressed gene modules that suggest functional networks

  • Track expression changes during development or disease progression

• Spatial transcriptomics integration:

  • Map FAM194A expression to specific anatomical regions

  • Correlate with cell type markers and functional tissue domains

  • Identify potential paracrine signaling relationships

• Single-cell proteomics approaches:

  • Develop antibody panels for mass cytometry including FAM194A

  • Perform imaging mass cytometry on tissue sections to preserve spatial context

  • Combine with functional readouts of cellular activity

• Integrative analysis methods:

  • Correlate FAM194A expression with gene networks and pathways

  • Perform trajectory analysis to map FAM194A expression during cellular differentiation

  • Integrate with epigenomic data to understand regulatory mechanisms

This approach has proven valuable for related FAM proteins, where gene correlation data from single-cell transcriptomics revealed associations with specific cellular components and pathways, including microtubule structures, mitochondria, and PCP-pathway associations .