Recombinant Human Protein FAM55B (FAM55B)

What is FAM55B protein and what is its relation to the NXPE family?

FAM55B, also known as NXPE2 (Uniprot ID: Q96DL1), is a member of the FAM55 protein family. It shares structural similarities with FAM55D (NXPE4), which belongs to the neurexophilin and PC-esterase domain family . Both proteins are part of a broader family of proteins with potential roles in cellular signaling and protein interactions, though their precise functions remain to be fully characterized through targeted research approaches.

What are the optimal storage and handling conditions for recombinant FAM55B?

Recombinant FAM55B should be stored at -20°C to -80°C, with a shelf life of approximately 12 months in lyophilized form and 6 months in liquid form . For working with the protein, it is recommended to reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, adding 5-50% glycerol (final concentration) before aliquoting for long-term storage . Repeated freeze-thaw cycles should be avoided to maintain protein integrity, and working aliquots can be stored at 4°C for up to one week .

How does the purity of recombinant FAM55B impact experimental outcomes?

The standard purity for research-grade recombinant FAM55B is >85% as determined by SDS-PAGE , which is comparable to the >80% purity standard for related proteins like FAM55D . This level of purity is suitable for most research applications, but higher purity may be required for specialized applications such as structural studies or when background contamination could interfere with sensitive assays. Always validate protein purity with both SDS-PAGE and functional assays before proceeding with critical experiments.

How should I design quasi-experimental approaches to study FAM55B function?

When designing quasi-experimental approaches for FAM55B research, consider family-based designs that can strengthen causal inferences about its biological role . These designs use "natural experiments" based on family relationships to test competing causal hypotheses . For cellular studies, implement controlled conditions where FAM55B expression is manipulated while maintaining physiologically relevant contexts. Such approaches are particularly valuable when random assignment is not possible but causal inferences are still sought .

What controls are essential when conducting blocking experiments with FAM55B antibodies?

For antibody validation and blocking experiments with FAM55B, follow similar protocols to those established for related proteins like FAM55D . Include a recombinant protein control fragment at approximately 100x molar excess based on concentration and molecular weight . Pre-incubate the antibody-protein control fragment mixture for 30 minutes at room temperature before proceeding with experiments . Essential controls include: (1) antibody alone, (2) recombinant protein alone, (3) the antibody-protein mixture, and (4) a non-specific protein control to confirm blocking specificity.

How can I implement genetic approaches to understand FAM55B function?

To implement genetic approaches for FAM55B functional studies, consider both family-based designs and molecular genetic testing methods such as Transmission Disequilibrium Tests . These approaches can help identify whether specific FAM55B variants are associated with particular phenotypes while controlling for population stratification . For cellular studies, CRISPR-Cas9 editing can provide precise genetic manipulation to determine protein function through loss-of-function or gain-of-function experiments in relevant cell types.

What is the recommended protocol for reconstituting and preparing FAM55B for experimental use?

To reconstitute lyophilized recombinant FAM55B:

• Briefly centrifuge the vial to bring contents to the bottom

• Add deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for stability

• Aliquot and store at -20°C for long-term use

For experimental applications, thaw aliquots at room temperature and keep on ice during handling. Avoid repeated freeze-thaw cycles by preparing single-use aliquots. For functional assays, equilibrate the protein in the appropriate buffer system that maintains protein stability and activity.

How do expression systems impact the properties of recombinant FAM55B?

Recombinant FAM55B is typically expressed in E. coli systems , similar to related proteins like FAM55D . This bacterial expression system offers advantages in yield and cost-effectiveness but lacks eukaryotic post-translational modifications. When designing experiments, consider whether these modifications might be essential for your specific research questions. Alternative expression systems such as insect cells or mammalian cells may be considered if native post-translational modifications are critical for function, though this should be balanced against potentially lower yields and higher production costs.

What methodological approaches can resolve contradictory findings in FAM55B research?

To resolve contradictory findings in FAM55B research, implement a multi-faceted methodological approach:

• Family-based, quasi-experimental designs to distinguish between genetic and environmental influences

• Multiple complementary techniques (e.g., biochemical, cellular, and genetic approaches) to validate findings

• Systematic variation of experimental conditions to identify context-dependent effects

• Quantitative comparison across experimental parameters using standardized metrics

This approach aligns with recommendations for resolving causal relationships in complex biological systems, where observational studies alone may not adequately test alternative hypotheses .

How can I distinguish between genetic and environmental factors affecting FAM55B function?

To distinguish between genetic and environmental factors affecting FAM55B function, implement family-based, quasi-experimental designs as described in critical research methodologies . These approaches include sibling comparison studies, offspring of twins or siblings designs, and in vitro fertilization designs that can rigorously examine causal inferences by testing competing hypotheses . When conducting cellular experiments, use controlled environmental manipulations with genetically identical cells to isolate environmental effects on FAM55B expression and function.

What experimental approaches can reveal molecular interactions between FAM55B and other proteins?

To investigate FAM55B's molecular interactions, employ a systematic approach combining:

• Affinity purification followed by mass spectrometry to identify potential binding partners

• Co-immunoprecipitation experiments to confirm direct interactions in cellular contexts

• Proximity ligation assays to visualize interactions in situ

• Pull-down assays using recombinant FAM55B as bait

• Surface plasmon resonance or isothermal titration calorimetry for quantitative binding measurements

These approaches should be complemented by functional validation studies to confirm the biological relevance of identified interactions.

How can I compare the functional properties of FAM55B (NXPE2) with FAM55D (NXPE4)?

To systematically compare these proteins, design parallel experiments examining:

• Expression patterns across tissues and cell types

• Binding partner profiles through affinity purification-mass spectrometry

• Functional effects of overexpression or knockdown in relevant cell lines

• Domain-specific functions through truncation or mutation studies

What statistical approaches are appropriate for analyzing FAM55B expression data across experimental conditions?

When analyzing FAM55B expression data, implement statistical approaches based on experimental design principles from quantitative research2. For controlled experiments comparing multiple conditions, use ANOVA with appropriate post-hoc tests, ensuring adequate statistical power. For complex experimental designs involving potential genetic confounding, more sophisticated statistical models may be necessary as highlighted in family-based research approaches . Always:

• Test data for normality and homogeneity of variance

• Consider repeated measures designs when appropriate

• Include correction for multiple comparisons

• Report effect sizes alongside p-values

• Provide transparent data visualization that represents both central tendency and variation

How should I interpret seeming contradictions between in vitro and in vivo findings about FAM55B?

To interpret contradictions between in vitro and in vivo findings, employ a structured analytical approach:

• Examine methodological differences that might explain discrepancies, including:

  • Expression levels (physiological vs. overexpression)

  • Cell/tissue context (isolated cells vs. intact systems)

  • Temporal factors (acute vs. chronic effects)

  • Species differences in protein structure or function

• Design bridging experiments that systematically vary conditions between the in vitro and in vivo extremes

• Consider implementing quasi-experimental designs to test competing hypotheses about causal mechanisms

• Develop integrated models that can accommodate conditional effects based on cellular or physiological context

What approaches can help determine if FAM55B findings are generalizable across different experimental systems?

To determine generalizability of FAM55B findings, implement a systematic cross-validation strategy:

• Test in multiple cell lines representing different tissue origins

• Compare results across species when possible

• Validate with different methodological approaches (e.g., knockdown, overexpression, domain mutations)

• Consider the limitations of specialized systems like IVF models, which may have generalizability concerns

• Implement family-based quasi-experimental designs when possible, as these can provide stronger evidence for causal relationships while maintaining external validity

What are common challenges in expressing and purifying recombinant FAM55B, and how can they be addressed?

Common challenges in recombinant FAM55B expression and purification include:

How can I troubleshoot non-specific binding when using anti-FAM55B antibodies?

To troubleshoot non-specific binding with anti-FAM55B antibodies:

• Validate antibody specificity using recombinant FAM55B as a positive control

• Implement blocking experiments with recombinant protein at 100x molar excess

• Optimize blocking conditions (time, temperature, blocking agent)

• Increase washing stringency (higher salt concentration, more washes)

• Pre-adsorb antibodies against tissues or lysates lacking FAM55B expression

• Test multiple antibodies targeting different epitopes

• For critical experiments, validate findings with orthogonal approaches not relying on antibodies

What approaches can overcome detection challenges for low-abundance FAM55B in complex biological samples?

To enhance detection of low-abundance FAM55B:

• Implement sample enrichment strategies:

  • Immunoprecipitation to concentrate the protein

  • Subcellular fractionation to reduce sample complexity

  • Selective precipitation techniques

• Employ signal amplification methods:

  • Tyramide signal amplification for immunohistochemistry

  • Enhanced chemiluminescence for Western blotting

  • Amplified ELISA systems

• Utilize high-sensitivity detection technologies:

  • Selected reaction monitoring mass spectrometry

  • Droplet digital PCR for transcript quantification

  • Super-resolution microscopy for localization studies

• Incorporate internal standards and calibration curves to quantify low levels accurately