Recombinant Human Protein FAM156A (FAM156A)

What is FAM156A and how is it classified structurally?

FAM156A (Family With Sequence Similarity 156, Member A) is classified as a transmembrane protein (TP) that spans the entire biological membrane. It belongs to the FAM156 subfamily and is also known as TMEM29 (Transmembrane protein 29/29B) . As a transmembrane protein, FAM156A contains hydrophobic regions that allow it to traverse the lipid bilayer of cell membranes. It is characterized as a polytopic protein, meaning it has multiple membrane-spanning domains . The protein's structure enables it to function as a gateway between the intracellular and extracellular environments, potentially regulating the transport of specific substances across the membrane barrier.

Unlike soluble proteins, FAM156A and other transmembrane proteins exhibit specific physicochemical properties that affect their experimental handling. They aggregate and precipitate in water, requiring specialized extraction methods using detergents or nonpolar solvents . This characteristic significantly influences experimental design considerations when working with this protein in laboratory settings.

What methodologies are recommended for detecting endogenous FAM156A in tissue samples?

For detecting endogenous FAM156A in tissue samples, researchers should employ multiple complementary approaches to ensure reliable results. Immunohistochemistry (IHC) using validated antibodies against human FAM156A represents a primary approach . When performing IHC, it is critical to include appropriate positive and negative controls to confirm specificity of staining patterns.

Western blotting provides a quantitative method for FAM156A detection, though specialized protocols for membrane proteins must be followed. These include:

• Optimized membrane protein extraction using appropriate detergents

• Extended transfer times at lower voltages to ensure complete transfer of hydrophobic proteins

• Use of validated anti-FAM156A antibodies with demonstrated specificity

• Inclusion of appropriate loading controls specific for membrane proteins

For transcript-level detection, quantitative PCR or RNA sequencing can be employed, though protein-level validation is still necessary due to potential post-transcriptional regulation. When analyzing expression patterns across multiple tissues, researchers should consider the methodological approaches used in developmental gene expression studies, which classify genes into distinct expression patterns (such as PTN1-PTN27) based on their temporal regulation .

How should researchers design experiments to study FAM156A function in cellular systems?

When designing experiments to investigate FAM156A function, researchers must account for its transmembrane nature and implement appropriate controls. A methodologically sound approach includes:

• Model system selection: Choose cell lines that naturally express FAM156A for knockdown studies or cells with minimal expression for overexpression experiments. Primary cells from relevant tissues may provide more physiologically relevant contexts .

• Gene manipulation strategy:

  • For transient suppression: siRNA or shRNA with validated target sequences

  • For stable knockout: CRISPR-Cas9 targeting conserved exons

  • For overexpression: Expression vectors with appropriate tags that don't interfere with transmembrane domains

• Experimental controls:

  • Include scrambled siRNA controls for knockdown experiments

  • Use empty vector transfections as controls for overexpression studies

  • Perform rescue experiments to confirm phenotype specificity

  • Include positive controls for expected cellular processes

• Functional readouts: Depending on the research question, measure:

  • Membrane transport activity using fluorescent substrates

  • Protein-protein interactions using proximity ligation assays

  • Subcellular localization through immunofluorescence or fractionation

  • Cellular phenotypes (proliferation, migration, differentiation)

• Experimental design principles: Implement randomization, appropriate replication (minimum three biological replicates), blinding where applicable, and proper statistical analysis .

What are the key considerations when extracting and purifying recombinant FAM156A protein?

Extracting and purifying transmembrane proteins like FAM156A presents significant challenges due to their hydrophobic nature. Researchers should consider the following methodological approaches:

• Expression system selection: Mammalian or insect cell expression systems often yield better results than bacterial systems for human transmembrane proteins. These eukaryotic systems provide appropriate post-translational modifications and membrane insertion machinery.

• Membrane protein extraction:

  • FAM156A will aggregate and precipitate in water, necessitating detergent-based extraction

  • Initial screening of multiple detergents (CHAPS, DDM, Triton X-100) to identify optimal solubilization conditions

  • Careful temperature control during extraction to prevent protein denaturation

• Purification strategy:

  • Affinity chromatography using appropriate tags (His, FLAG, etc.)

  • Size exclusion chromatography to separate protein complexes

  • Consider detergent exchange during purification to improve stability

• Quality control measures:

  • SDS-PAGE and Western blotting to confirm purity and identity

  • Circular dichroism to verify secondary structure integrity

  • Dynamic light scattering to assess homogeneity and aggregation state

• Functional validation: Develop assays to confirm that purified protein retains native activity, potentially through reconstitution into liposomes or nanodiscs to test transport function.

How can researchers reconcile contradictory findings about FAM156A in the literature?

When faced with contradictory findings regarding FAM156A, researchers should implement a systematic approach to resolve discrepancies:

• Methodological replication: Reproduce original experiments using identical methods, paying particular attention to:

  • Cell lines and culture conditions

  • Reagent sources and validation status

  • Experimental protocols and analysis methods

• Cross-validation using multiple techniques:

  • Apply complementary methodologies to address the same question

  • Use both protein and transcript-level measurements

  • Implement both in vitro and in vivo approaches where feasible

• Variables that may explain contradictions:

  • Cell type-specific effects: Test multiple relevant cell lines

  • Splice variant expression: Verify which isoforms are being detected

  • Antibody specificity: Validate antibodies using knockout controls

  • Experimental conditions: Systematically vary conditions to identify critical parameters

• Meta-analysis approach:

  • Systematically analyze all available data

  • Weight findings based on methodological quality

  • Identify patterns across studies despite individual variations

• Collaborative verification:

  • Engage multiple laboratories in standardized studies

  • Share reagents and protocols to ensure consistency

  • Perform blinded analyses to eliminate bias

What methods are most effective for studying FAM156A protein-protein interactions?

Investigating protein-protein interactions involving transmembrane proteins like FAM156A requires specialized approaches:

• Membrane-specific yeast two-hybrid systems:

  • Split-ubiquitin membrane yeast two-hybrid (MYTH)

  • G-protein fusion yeast two-hybrid

  These modified systems allow screening for interactions in membrane environments, overcoming limitations of conventional Y2H for transmembrane proteins.

• Proximity-based labeling approaches:

  • BioID or TurboID fusion proteins to label proximal proteins

  • APEX2-based proximity labeling in living cells

  • Mass spectrometric identification of labeled proteins

• Co-immunoprecipitation with membrane solubilization:

  • Optimize detergent conditions to maintain interactions

  • Use crosslinking to stabilize transient interactions

  • Employ antibodies validated for immunoprecipitation efficiency

• Fluorescence-based interaction studies:

  • Fluorescence resonance energy transfer (FRET)

  • Bioluminescence resonance energy transfer (BRET)

  • Split fluorescent protein complementation assays

The following table summarizes the advantages and limitations of each approach:

How can researchers determine the role of FAM156A in development and disease progression?

Understanding FAM156A's role in development and disease requires integrated experimental approaches:

• Expression pattern analysis:

  • Profile FAM156A expression across developmental stages and tissues

  • Classify expression patterns similar to the methodology used in lung development studies (PTN1-PTN27)

  • Determine if FAM156A follows patterns like decreasing expression during development (PTN1) or increasing expression (PTN27)

• Loss-of-function studies:

  • Generate conditional knockout models to study tissue-specific functions

  • Use temporal control of gene deletion to identify developmental windows of activity

  • Analyze resulting phenotypes across multiple organ systems

• Disease association studies:

  • Compare FAM156A expression between normal and diseased tissues

  • Apply prognostic value assessment methods similar to those used in lung adenocarcinoma studies

  • Correlate expression with clinical parameters and disease progression

• Mechanistic investigations:

  • Identify downstream molecular changes following FAM156A manipulation

  • Map signaling pathways affected by FAM156A alterations

  • Determine if FAM156A alterations affect fundamental cellular processes

• Translational implications:

  • Assess potential as a biomarker based on expression patterns

  • Evaluate as a therapeutic target if disease associations are established

  • Develop intervention strategies based on mechanistic insights

What genomic and epigenetic approaches can reveal FAM156A regulation mechanisms?

Understanding FAM156A gene regulation requires comprehensive genomic and epigenetic analyses:

• Promoter characterization:

  • In silico identification of transcription factor binding sites

  • Chromatin immunoprecipitation (ChIP) to identify proteins binding to the FAM156A promoter

  • Reporter assays to validate promoter activity and responsive elements

• Epigenetic profiling:

  • DNA methylation analysis using bisulfite sequencing

  • Histone modification mapping using ChIP-seq

  • Chromatin accessibility analysis using ATAC-seq or DNase-seq

• Long-range interaction analysis:

  • Chromosome Conformation Capture (3C) and derivatives (4C, 5C, Hi-C)

  • Identify enhancers that regulate FAM156A expression

  • Map topologically associating domains (TADs) containing the FAM156A locus

• Transcriptional regulation studies:

  • Identify transcription factors controlling FAM156A using perturbation studies

  • Validate binding using electrophoretic mobility shift assays

  • Confirm functional impact through site-directed mutagenesis

• Post-transcriptional regulation:

  • miRNA targeting prediction and validation

  • RNA-binding protein identification through RNA immunoprecipitation

  • mRNA stability assessments following regulatory factor manipulation

How can researchers overcome difficulties in detecting low-abundance FAM156A protein?

Detecting low-abundance transmembrane proteins like FAM156A presents unique challenges. Methodological solutions include:

• Enhanced extraction protocols:

  • Optimize membrane protein extraction with specialized detergents

  • Implement sequential extraction to increase yield

  • Use phospholipid nanodiscs to maintain native conformation

• Signal amplification methods:

  • Utilize tyramide signal amplification for immunohistochemistry

  • Implement proximity ligation assays for improved sensitivity

  • Apply mass spectrometry with targeted multiple reaction monitoring (MRM)

• Enrichment strategies:

  • Perform subcellular fractionation to concentrate membrane proteins

  • Use immunoprecipitation with validated antibodies

  • Consider lectin affinity for glycosylated forms of FAM156A

• Optimized Western blot protocol:

  • Transfer conditions tailored for transmembrane proteins (longer time, lower voltage)

  • Specialized blocking to reduce background (e.g., 5% BSA instead of milk proteins)

  • Extended primary antibody incubation at 4°C

  • Enhanced chemiluminescence detection systems with longer exposure times

• Validation strategies:

  • Include positive controls from tissues known to express FAM156A

  • Use recombinant protein standards for quantification

  • Verify signal specificity using genetic knockdown approaches

What are common artifacts in FAM156A immunolocalization studies and how can they be avoided?

Immunolocalization studies of transmembrane proteins like FAM156A are prone to artifacts. Prevention strategies include:

• Fixation optimization:

  • Test multiple fixatives (paraformaldehyde, glutaraldehyde, methanol)

  • Optimize fixation duration and temperature

  • Consider specialized fixation for membrane proteins (e.g., glyoxal)

• Antibody validation:

  • Verify antibody specificity using knockout/knockdown controls

  • Test multiple antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

• Permeabilization considerations:

  • Optimize detergent type and concentration for membrane access

  • Balance permeabilization efficiency with epitope preservation

  • Consider non-detergent permeabilization methods for sensitive epitopes

• Signal specificity controls:

  • Include secondary-only controls to assess non-specific binding

  • Use isotype controls to evaluate background staining

  • Perform parallel studies with fluorescent protein-tagged constructs

• Image acquisition and analysis:

  • Capture images using identical settings across samples

  • Implement blinded analysis to prevent bias

  • Use quantitative analysis methods with appropriate thresholding

What are effective strategies for validating FAM156A knockdown or overexpression models?

Proper validation of genetic manipulation models is crucial for reliable FAM156A functional studies:

• Transcript-level validation:

  • qRT-PCR with primers targeting multiple exons

  • RNA-seq to assess global expression changes

  • Northern blotting for comprehensive isoform analysis

• Protein-level confirmation:

  • Western blotting with validated antibodies

  • Immunofluorescence to assess cellular localization

  • Flow cytometry for quantitative single-cell analysis

• Genomic validation for knockout models:

  • PCR genotyping with primers flanking the targeted region

  • Sequencing to confirm precise modifications

  • Analysis of potential off-target effects

• Functional validation:

  • Rescue experiments to confirm phenotype specificity

  • Dose-response assessment in inducible systems

  • Evaluation of known downstream effects

• Controls and standards:

  • Include positive and negative controls in all validation experiments

  • Establish minimum validation criteria before proceeding with functional studies

  • Document all validation results comprehensively

What emerging technologies could advance understanding of FAM156A function?

Several cutting-edge technologies show promise for elucidating FAM156A function:

• Structural biology approaches:

  • Cryo-electron microscopy for membrane protein structures

  • AlphaFold and other AI-based structure prediction methods

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Advanced genome editing:

  • Base editing for introducing specific mutations without double-strand breaks

  • Prime editing for precise genetic modifications

  • CRISPR activation/inhibition for modulating expression without genetic alteration

• Single-cell technologies:

  • Single-cell RNA-seq to identify cell populations expressing FAM156A

  • Single-cell proteomics to quantify protein levels across heterogeneous populations

  • Spatial transcriptomics to map expression in tissue contexts

• Advanced imaging:

  • Super-resolution microscopy for nanoscale localization

  • Live-cell imaging with improved fluorescent tags

  • Correlative light and electron microscopy for ultrastructural context

• Organoid and advanced cell models:

  • Patient-derived organoids for disease modeling

  • Microphysiological systems (organ-on-chip) for functional studies

  • Advanced differentiation protocols to study developmental roles

How might FAM156A research contribute to understanding broader biological processes?

FAM156A research has potential implications for multiple fundamental biological processes:

• Membrane biology:

  • Understanding transmembrane protein trafficking and localization

  • Elucidating mechanisms of membrane transport regulation

  • Revealing membrane domain organization principles

• Developmental biology:

  • Clarifying roles of membrane proteins in tissue differentiation

  • Understanding temporal regulation of gene expression during development

  • Identifying novel developmental signaling mechanisms

• Disease mechanisms:

  • Potential roles in diseases with membrane transport defects

  • Applications of prognostic gene signatures in cancer, similar to those identified in lung adenocarcinoma studies

  • Understanding membrane protein dysfunction in pathological conditions

• Evolutionary biology:

  • Comparative analysis of membrane protein function across species

  • Understanding evolutionary conservation of transmembrane domains

  • Elucidating specialized adaptations of membrane proteins

• Systems biology:

  • Integration of FAM156A into broader protein interaction networks

  • Understanding coordinated regulation of membrane protein families

  • Modeling membrane protein dynamics in cellular systems

What interdisciplinary approaches could accelerate FAM156A research?

Advancing FAM156A research will benefit from interdisciplinary collaboration:

• Computational-experimental integration:

  • Predictive modeling of protein structure and function

  • Machine learning approaches for pattern recognition in expression data

  • Simulation of membrane protein dynamics

• Clinical-basic science partnerships:

  • Translating fundamental findings to clinical applications

  • Accessing patient samples for validation studies

  • Identifying disease-relevant research questions

• Physics-biology interface:

  • Biophysical characterization of membrane protein properties

  • Advanced spectroscopy for structural analysis

  • Nanoscale measurements of protein-membrane interactions

• Chemistry-biology collaboration:

  • Development of small molecule modulators of FAM156A

  • Novel labeling strategies for detection and tracking

  • Improved methods for membrane protein solubilization

• Engineering-biology approaches:

  • Biosensor development for functional assays

  • Microfluidic systems for high-throughput screening

  • Synthetic biology tools for studying protein function