Recombinant Human Protein FAM168B (FAM168B)

What is FAM168B protein and what are its primary biological functions?

FAM168B (Family with Sequence Similarity 168 Member B), also known as KIAA0280L, MANI, Myelin-associated neurite-outgrowth inhibitor, or p20, functions primarily as an inhibitor of neuronal axonal outgrowth. At the molecular level, it acts as a negative regulator of CDC42 and STAT3 signaling pathways while positively regulating STMN2 and CDC27 . The protein plays a significant role in neuronal development and axonal growth regulation by modulating these key signaling pathways.

The protein is predominantly expressed in brain tissue and is localized to the axon, perinuclear region of cytoplasm, and plasma membrane . This subcellular distribution pattern aligns with its function in regulating neuronal development and axonal outgrowth. The characteristic domain structure includes the conserved FAM168A/MANI family domain, which is critical for its inhibitory function in neurite outgrowth.

What are the recommended methods for detecting FAM168B expression in tissue samples?

For detecting FAM168B expression in tissue samples, immunohistochemistry on paraffin-embedded sections (IHC-P) and immunocytochemistry/immunofluorescence (ICC/IF) are proven effective techniques. When performing IHC-P, a 1/100 dilution of anti-FAM168B antibody (such as ab238117) has been successfully used on human skeletal muscle and gastric cancer tissues .

For ICC/IF applications, the same 1/100 antibody dilution has demonstrated excellent results in human cell lines including PC-3 (prostate adenocarcinoma) and A549 (lung carcinoma) cells, with detection using secondary Alexa-Fluor®488-conjugated goat anti-rabbit IgG .

The protocol for these applications typically involves:

• Fixation of tissue samples or cells

• Antigen retrieval (for paraffin sections)

• Blocking of non-specific binding

• Overnight incubation with primary antibody at 4°C

• Washing and application of appropriate secondary antibody

• Counterstaining and mounting

For optimal results, researchers should include positive control tissues known to express FAM168B (such as brain tissue) and negative controls with isotype-matched IgG to validate specificity.

How should recombinant FAM168B protein be reconstituted and stored for maximum stability?

For optimal stability and activity of recombinant FAM168B protein, follow these methodological steps:

• Centrifuge the vial briefly before opening to bring contents to the bottom.

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

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

• Aliquot the reconstituted protein to minimize freeze-thaw cycles.

• Store aliquots at -20°C/-80°C for long-term storage .

Working aliquots should be prepared in volumes appropriate for single experiments to avoid repeated handling of the stock solution. For functional assays, it's recommended to perform a dose-response assessment to determine the optimal working concentration for each specific application.

How can I design experiments to investigate FAM168B's role in neurite outgrowth inhibition?

To investigate FAM168B's role in neurite outgrowth inhibition, consider implementing these methodological approaches:

Gain and Loss of Function Studies:

• CRISPR-Cas9 or shRNA knockdown of FAM168B in neuronal cell lines or primary neurons.

• Overexpression of FAM168B using lentiviral or plasmid vectors.

• Expression of truncated variants to identify functional domains essential for inhibitory activity.

Functional Assays:

• Neurite outgrowth measurements using time-lapse microscopy with automated image analysis.

• Co-culture experiments with FAM168B-expressing cells and neurons to assess contact-dependent inhibition.

• Growth cone collapse assays to evaluate acute effects on axonal dynamics.

Molecular Pathway Analysis:

• Phosphorylation state analysis of STAT3 in response to FAM168B manipulation.

• CDC42 activity assays using pull-down techniques with GST-PAK1 binding domain.

• Co-immunoprecipitation experiments to confirm direct interactions with CDC42, STAT3, and STMN2.

When designing these experiments, it's critical to include appropriate controls and establish baseline measurements. Statistical analysis should account for the inherent variability in neuronal cultures by using sufficient biological replicates (minimum n=3) and appropriate statistical tests based on data distribution . The inclusion of positive controls (known inhibitors like NogoA) and negative controls will strengthen the validity of your findings.

What approaches can be used to study the interaction between FAM168B and its regulatory targets CDC42 and STAT3?

To investigate interactions between FAM168B and its regulatory targets CDC42 and STAT3, implement these specialized approaches:

Co-immunoprecipitation (Co-IP) and Pull-down Assays:

• Perform Co-IP using anti-FAM168B antibodies in neuronal cell lysates to capture protein complexes.

• Use recombinant His-tagged FAM168B protein as bait in pull-down assays with cell lysates.

• Analyze precipitated complexes by western blotting using antibodies against CDC42 and STAT3.

Proximity Ligation Assay (PLA):

• Apply PLA in fixed cells or tissue sections to visualize direct protein-protein interactions in situ.

• Quantify interaction signals in different subcellular compartments to determine localization patterns.

FRET/BRET Analysis:

• Generate fluorescent protein fusions of FAM168B and CDC42/STAT3.

• Measure energy transfer in live cells to assess proximity and dynamic interactions.

Functional Domain Mapping:

• Create a series of truncated FAM168B constructs to identify interaction domains.

• Perform site-directed mutagenesis of conserved residues within the FAM168A/MANI domain.

• Assess binding affinities using surface plasmon resonance or microscale thermophoresis.

Activity Assays:

• Measure CDC42 GTPase activity in the presence of varying concentrations of recombinant FAM168B.

• Assess STAT3 phosphorylation and nuclear translocation following FAM168B overexpression or knockdown.

These techniques should be applied in complementary fashion to establish both physical interactions and functional consequences. When designing interaction studies, it's important to consider that these interactions may be transient or dependent on specific cellular contexts or signaling states .

What are common challenges when working with recombinant FAM168B protein and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant FAM168B protein. Here are methodological solutions to address these issues:

Protein Solubility Issues:

• When reconstituting lyophilized FAM168B, start with a lower concentration (0.1 mg/mL) and gradually increase if needed.

• Consider adding non-ionic detergents (0.01-0.05% Tween-20) to prevent aggregation.

• Use freshly prepared buffers and filter solutions to remove any particulates.

Loss of Activity During Storage:

• Add stabilizing agents such as 1-5 mM DTT or 5-10% glycerol to maintain protein integrity.

• Store at -80°C in small aliquots to minimize freeze-thaw cycles.

• Monitor protein stability by SDS-PAGE before each critical experiment.

Inconsistent Experimental Results:

• Standardize protein concentration determination methods (BCA or Bradford assay).

• Validate protein activity using consistent positive controls in functional assays.

• Ensure consistent handling between experiments, including thawing procedures and temperature conditions.

Protein Degradation:

• Add protease inhibitors to working solutions (PMSF, EDTA, or commercial cocktails).

• Keep protein samples on ice during handling and avoid prolonged incubation at room temperature.

• Verify protein integrity by western blot before functional experiments.

Optimizing Functional Assays:

• Perform dose-response experiments to determine the optimal protein concentration.

• Include appropriate time course analyses to capture both immediate and delayed effects.

• When studying inhibitory effects, pre-incubate cells with the protein before stimulation with activating factors.

Thorough documentation of experimental conditions and systematic approach to troubleshooting will help identify the source of variability in experiments using recombinant FAM168B protein .

How should I analyze and interpret data from FAM168B knockdown or overexpression experiments?

When analyzing data from FAM168B knockdown or overexpression experiments, implement these methodological approaches:

Verification of Manipulation Efficiency:

• Quantify FAM168B mRNA levels using qRT-PCR with appropriate reference genes.

• Confirm protein level changes by western blot with densitometric analysis.

• Document efficiency across multiple experiments to establish consistency.

Phenotypic Analysis:

• For neurite outgrowth studies, measure multiple parameters including:

  • Total neurite length per cell

  • Number of primary neurites

  • Branching complexity

  • Growth cone area

• Use automated image analysis software to reduce bias and increase throughput.

• Analyze at least 100 cells per condition across 3+ independent experiments.

Statistical Approach:

• Select appropriate statistical tests based on data distribution:

  • For normally distributed data: t-tests (two groups) or ANOVA (multiple groups)

  • For non-parametric data: Mann-Whitney U or Kruskal-Wallis tests

• Control for multiple comparisons using Bonferroni or Tukey's methods.

• Report effect sizes alongside p-values to indicate biological significance.

Molecular Pathway Analysis:

• Examine changes in downstream targets (CDC42, STAT3, STMN2) at both mRNA and protein levels.

• Incorporate phospho-specific antibodies to assess activation states of signaling molecules.

• Consider rescue experiments to confirm specificity of observed effects.

Control Considerations:

• Include appropriate mock-transfected and scrambled/empty vector controls.

• Assess potential off-target effects by examining related family members.

• Monitor cell health markers to distinguish specific effects from generalized toxicity.

When interpreting results, remember that variability in biological systems is expected. Focus on consistent trends across replicates rather than isolated observations, and consider how experimental variables such as cell density and culture conditions might influence results . Maintaining consistent experimental conditions across all groups will help reduce unsystematic variability and increase sensitivity to detect treatment effects.

How conserved is FAM168B across species and what can comparative studies tell us about its function?

FAM168B demonstrates notable evolutionary conservation across vertebrate species, providing valuable insights into its fundamental biological functions. Comparative analysis reveals:

The FAM168A/MANI domain (IPR029247) represents the most highly conserved region across species, suggesting its critical importance to protein function . This conservation extends from humans to zebrafish, indicating an evolutionarily ancient role in neuronal development.

Cross-Species Comparison Data:

In zebrafish (Danio rerio), FAM168B is expressed in the brain, mirroring its expression pattern in mammals . This conservation of tissue-specific expression further supports a conserved neurological function. Functional studies across species have demonstrated that the inhibitory effect on neurite outgrowth is preserved, suggesting this represents an ancient mechanism for regulating neuronal development.

When designing comparative studies, researchers should focus on:

• Cross-species rescue experiments to test functional equivalence

• Domain swapping between orthologs to identify species-specific regulatory regions

• Comparing interaction profiles with conserved binding partners (CDC42, STAT3)

• Examining expression patterns during development across different model organisms

These comparative approaches can reveal both the core conserved functions of FAM168B and any species-specific adaptations that have evolved, providing deeper insights into its fundamental biological roles and potential relevance to neurological disorders .

What is known about FAM168B expression patterns in different tissues and developmental stages?

Current research indicates that FAM168B exhibits specific spatiotemporal expression patterns that provide important insights into its biological functions:

Tissue-Specific Expression:
FAM168B is predominantly expressed in neural tissues, with highest expression levels observed in the brain . This neuronal enrichment aligns with its functional role as a regulator of neurite outgrowth and axonal development. Secondary expression has been documented in skeletal muscle tissue, suggesting potential additional functions in non-neuronal contexts .

Developmental Regulation:
Expression analysis across developmental stages reveals dynamic regulation of FAM168B:

• In zebrafish models, FAM168B expression is detected during early neurogenesis and increases as neural circuits form .

• The protein's inhibitory effects on neurite outgrowth suggest it may play a critical role in fine-tuning neural circuit development by preventing excessive axonal growth or promoting appropriate pruning.

Subcellular Localization:
Within cells, FAM168B demonstrates specific compartmentalization:

• Axonal localization - consistent with its role in regulating axonal growth

• Perinuclear region of cytoplasm - suggesting potential involvement in transcriptional regulation

• Plasma membrane association - indicating direct interaction with membrane-associated signaling components

Experimental Approaches for Expression Analysis:
To effectively characterize FAM168B expression patterns, researchers should consider:

• Single-cell RNA sequencing to identify specific neuronal subtypes expressing the protein

• Temporal analysis using developmental tissue series

• Combined in situ hybridization and immunohistochemistry to correlate mRNA and protein expression

• Reporter gene constructs to visualize expression in live tissues during development

These methodologies provide complementary data to build a comprehensive understanding of when and where FAM168B functions during normal development and in pathological states .