Recombinant Mouse Transmembrane protein FAM155B (Fam155b)

What is the molecular structure and characterization of FAM155B protein?

FAM155B (also known as NALF2, TED, or TMEM28) is a transmembrane protein consisting of 472 amino acids with a predicted molecular weight of 52.5 kDa and an isoelectric point of approximately 8.2. The protein contains two transmembrane domains and exhibits high expression in heart, thyroid, and brain tissues. Leucine (11.4%) and Proline (10%) are the most prominent amino acids in its composition . The protein belongs to a family whose function remains incompletely characterized by the scientific community, though its structure suggests membrane-associated functionality.

When working with recombinant versions, researchers should note that commercial preparations typically offer partial protein sequences with purity levels >85% as determined by SDS-PAGE analysis . For structural studies, it is essential to consider that there are two known protein isoforms: isoform 1 (340 amino acids) and isoform 2 (292 amino acids) .

What are the optimal storage conditions for maintaining FAM155B protein stability?

The stability of Recombinant Mouse Transmembrane protein FAM155B is influenced by multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself. For optimal preservation:

• Lyophilized form maintains stability for approximately 12 months when stored at -20°C/-80°C

• Liquid formulations generally maintain stability for up to 6 months at -20°C/-80°C

• Working aliquots can be stored at 4°C for up to one week

• Repeated freeze-thaw cycles should be strictly avoided to prevent protein degradation

For experiments requiring extended timeframes, researchers should prepare multiple small aliquots after reconstitution rather than storing a single stock solution to minimize freeze-thaw damage.

What is the recommended reconstitution protocol for lyophilized FAM155B protein?

To ensure optimal protein functionality, follow this methodological approach for reconstitution:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation for long-term storage)

• Prepare small aliquots for storage at -20°C/-80°C to minimize freeze-thaw cycles

This reconstitution approach provides the best balance between protein stability and experimental utility. The addition of glycerol serves as a cryoprotectant to minimize structural damage during freezing.

How can researchers verify the functional integrity of recombinant FAM155B after reconstitution?

Verification of functional integrity for FAM155B requires multiple analytical approaches:

• Primary structure confirmation: Peptide mass fingerprinting or N-terminal sequencing can verify protein identity compared to the expected sequence (UniProt accession A2BDP1) .

• Secondary/tertiary structure assessment: Circular dichroism spectroscopy can evaluate whether proper protein folding has been maintained after reconstitution, particularly important for transmembrane proteins.

• Functional assays: Since FAM155B (now also known as NALCN channel auxiliary factor 2) may function in ion channel regulation, patch-clamp electrophysiology or fluorescence-based ion flux assays in cells expressing NALCN channels can assess functional activity.

• Binding studies: Surface plasmon resonance or co-immunoprecipitation to verify interaction with known binding partners.

For rigorous experimental approaches, researchers should incorporate both positive controls (freshly prepared protein) and negative controls (heat-denatured protein) to establish baseline functional parameters.

What are the considerations for designing experiments involving FAM155B in neuronal tissue?

When designing experiments involving FAM155B in neuronal tissue, researchers should consider:

• Expression pattern analysis:

  • Verify endogenous expression levels in specific neuronal populations before introducing recombinant protein

  • Consider developmental timing, as expression may vary across developmental stages

• Experimental delivery methods:

  • For in vitro studies: Lipid-based transfection may be suboptimal due to FAM155B's transmembrane nature; consider viral vectors for neuronal expression

  • For in vivo studies: Stereotaxic injection of viral vectors expressing FAM155B may provide spatially controlled expression

• Functional readouts:

  • Electrophysiological recordings to assess changes in membrane potential or ion channel activity

  • Calcium imaging to detect alterations in neuronal signaling

  • Behavioral assays to evaluate potential effects on neural circuit function

• Controls and validation:

  • Include tagged versions (e.g., GFP-fusion) to track localization

  • Implement knockdown/knockout controls to establish baseline function

  • Use structure-function mutants to identify critical domains

The transmembrane nature of FAM155B presents unique experimental challenges that require careful consideration of membrane topology and potential disruption of native protein interactions.

What methodological approaches can address the challenges in differentiating between FAM155B isoforms in experimental systems?

Differentiating between the two known isoforms of FAM155B requires specialized methodological approaches:

For comprehensive isoform analysis, researchers should employ multiple complementary techniques. The 48-amino acid difference between isoforms may result in functional distinctions that could be relevant to experimental outcomes, particularly in studies focusing on protein-protein interactions or localization patterns.

How can researchers address solubility issues when working with recombinant FAM155B protein?

As a transmembrane protein, FAM155B presents inherent solubility challenges that researchers must address methodically:

• Buffer optimization strategies:

  • Test buffers with varying pH (6.5-8.5) to identify optimal solubility conditions

  • Incorporate mild detergents (0.01-0.1% Triton X-100 or 0.5-1% CHAPS) to maintain membrane protein solubility

  • Add stabilizing agents such as glycerol (5-10%) or specific divalent cations

• Temperature considerations:

  • Conduct solubility trials at different temperatures (4°C, room temperature, 37°C)

  • Implement gradual temperature transitions to prevent precipitation

• Protein concentration effects:

  • Titrate protein concentration, starting with lower concentrations (0.01-0.1 mg/mL)

  • Monitor aggregation using dynamic light scattering at various concentrations

• Co-solubilizing factors:

  • Consider adding specific lipids that might stabilize the native membrane environment

  • Test protein stabilizing compounds such as arginine or sucrose

When persistent solubility issues occur, researchers might need to explore protein engineering approaches, such as creating soluble domains or fusion constructs that maintain functional epitopes while improving handling characteristics.

What are the potential pitfalls in data interpretation when analyzing FAM155B function in experimental models?

Researchers analyzing FAM155B function should be aware of several potential pitfalls:

• Overexpression artifacts:

  • Artificially high expression levels may cause mislocalization or aberrant interactions

  • Solution: Titrate expression levels and compare with physiological expression patterns

  • Validate findings using knockin approaches maintaining endogenous regulation

• Truncation effects:

  • Partial recombinant proteins may lack crucial functional domains

  • Solution: Compare results with full-length protein when possible

  • Map functional domains through systematic deletion analysis

• Species-specific differences:

  • Mouse FAM155B may exhibit distinct properties from human orthologs

  • Solution: Perform comparative studies across species when interpreting translational relevance

  • Analyze sequence conservation in functional domains

• Contextual dependencies:

  • Function may vary across cell types due to different interaction partners

  • Solution: Study the protein in multiple relevant cell types

  • Identify cell-specific binding partners through proximity labeling approaches

• Technical limitations:

  • Antibody cross-reactivity with related family members

  • Solution: Validate antibody specificity using knockout controls

  • Employ multiple detection methods for confirmation

Careful experimental design with appropriate controls and validation across multiple methodological approaches can help mitigate these potential pitfalls.

How can FAM155B be effectively utilized in studies examining ion channel regulation?

Recent research indicates that FAM155B functions as NALCN channel auxiliary factor 2 (NALF2) , suggesting important roles in ion channel regulation. Researchers can leverage this in several ways:

• Electrophysiological studies:

  • Co-express FAM155B with NALCN channel components in heterologous systems

  • Compare channel kinetics and conductance properties with and without FAM155B

  • Perform structure-function analyses by mutating key residues in the transmembrane domains

• Protein-protein interaction characterization:

  • Map interaction domains between FAM155B and NALCN using truncation constructs

  • Employ proximity labeling techniques (BioID, APEX) to identify additional interacting partners

  • Perform co-immunoprecipitation studies under varying ionic conditions

• Physiological relevance:

  • Implement conditional knockout approaches in specific tissues

  • Develop FAM155B modulators to probe functional consequences

  • Investigate disease models with known ion channel dysregulation

• Biophysical approaches:

  • Reconstruct channel complexes in artificial lipid bilayers

  • Conduct single-particle cryo-EM to visualize channel complexes with FAM155B

  • Perform molecular dynamics simulations to predict conformational changes

These approaches can provide insights into the mechanisms by which FAM155B modulates ion channel function, potentially revealing new therapeutic targets for channelopathies.

What comparative approaches can reveal insights about FAM155B evolutionary conservation and functional significance?

Comparative approaches can yield valuable insights into FAM155B's core functions:

When implementing these approaches, researchers should:

• Focus on transmembrane domains, which likely represent functionally conserved regions

• Examine conservation patterns in relation to known channelopathies

• Correlate evolutionary conservation with tissue-specific expression patterns

• Consider the two isoforms separately in evolutionary analyses, as they may have distinct evolutionary trajectories

• Analyze selective pressure signatures to identify regions under positive or purifying selection

This evolutionary perspective can provide context for experimental findings and guide hypothesis generation regarding FAM155B's fundamental biological roles.

What emerging technologies might enhance our understanding of FAM155B function in complex tissues?

Several cutting-edge technologies show promise for advancing FAM155B research:

• Single-cell multi-omics:

  • Single-cell transcriptomics coupled with proteomics to map FAM155B expression

  • Spatial transcriptomics to visualize expression patterns within complex tissues

  • Single-cell ATAC-seq to identify regulatory elements controlling expression

• Advanced imaging techniques:

  • Super-resolution microscopy for nanoscale localization

  • Expansion microscopy to visualize protein interactions in intact tissue

  • Label-free imaging methods to track dynamic changes in native contexts

• Functional genomics approaches:

  • CRISPR interference/activation for temporal control of expression

  • Prime editing for precise introduction of clinically relevant mutations

  • Massively parallel reporter assays to identify regulatory elements

• Structural biology innovations:

  • Cryo-electron tomography for in situ structural analysis

  • Integrative structural biology combining multiple data types

  • AlphaFold2 predictions validated through targeted experimental approaches

• Organoid technologies:

  • Brain organoids to study function in developing neural tissues

  • Multi-organ-on-chip systems to examine tissue interactions

  • Patient-derived organoids for disease modeling

These technologies, applied in concert, can provide a more comprehensive understanding of FAM155B's role in normal physiology and disease states, potentially revealing new therapeutic strategies for conditions involving ion channel dysfunction.

What are the methodological considerations for studying FAM155B in the context of neurodevelopmental disorders?

Given FAM155B's expression in brain tissue and potential role in ion channel regulation, researchers investigating neurodevelopmental contexts should consider:

• Model system selection:

  • Appropriate animal models that recapitulate relevant aspects of neurodevelopment

  • Human iPSC-derived neural cells to capture species-specific functions

  • Transgenic models with conditional expression during specific developmental windows

• Temporal dynamics:

  • Implement inducible systems to manipulate expression at specific developmental stages

  • Utilize lineage tracing to track cells expressing FAM155B through development

  • Perform time-course analyses to identify critical periods

• Circuit-level analyses:

  • Combine electrophysiology with optogenetics to probe functional consequences

  • Implement connectomics approaches to assess circuit alterations

  • Use calcium imaging in intact circuits to evaluate signaling changes

• Translational considerations:

  • Correlate findings with human genetic data from neurodevelopmental disorders

  • Screen for compounds that modulate FAM155B function as potential therapeutic leads

  • Develop biomarkers for FAM155B dysregulation in accessible patient samples

These methodological approaches can help establish causal relationships between FAM155B dysfunction and specific neurodevelopmental phenotypes, potentially leading to new diagnostic or therapeutic strategies.