Recombinant Pongo abelii Regulator of microtubule dynamics protein 3 (FAM82A2)

What is FAM82A2 and what are its alternative names?

FAM82A2 (family with sequence similarity 82, member A2) is also known as Regulator of microtubule dynamics protein 3 (RMD-3). Additional synonyms include FAM82C, PTPIP51 (protein tyrosine phosphatase-interacting protein 51), FLJ10579, TCPTP interacting protein 51, and microtubule-associated protein. This protein is classified as a regulator of microtubule dynamics, suggesting its involvement in cytoskeletal organization and cellular structure maintenance .

What are the known protein interactions of FAM82A2?

FAM82A2 has been shown to interact with several proteins based on various detection methods including yeast two-hybrid, co-immunoprecipitation, and pull-down assays. Key interaction partners include:

• YWHAB and YWHAG (14-3-3 protein family members)

• PTPN1 (Protein tyrosine phosphatase non-receptor type 1)

• VAPA (Vesicle-associated membrane protein-associated protein A)

• Rmdn3 (Regulator of microtubule dynamics protein 3)

• PSEN1 (Presenilin-1)

• EGFL8 (EGF-like domain-containing protein 8)

These interactions suggest potential roles in cellular signaling, vesicular transport, and microtubule organization.

How should I design an experiment to study FAM82A2 function in vitro?

When designing experiments to study FAM82A2 function, follow these structured steps:

• Define your variables clearly:

  • Independent variable: Typically the experimental conditions you're manipulating (e.g., FAM82A2 concentration, presence of interaction partners)

  • Dependent variable: The outcome you're measuring (e.g., microtubule dynamics, binding affinity)

  • Control variables: Factors kept constant across all experimental conditions

• Formulate a specific, testable hypothesis about FAM82A2 function based on its known role as a regulator of microtubule dynamics

• Design experimental treatments:

  Treatment Group	FAM82A2 Concentration	Interaction Partner	Other Variables
  Control	0 nM	None	Standard buffer
  Low dose	10 nM	None	Standard buffer
  High dose	100 nM	None	Standard buffer
  Partner study	50 nM	YWHAB (50 nM)	Standard buffer

• Determine appropriate measurement methods for the dependent variable, such as microscopy for visualizing microtubule structures or binding assays for protein interactions

• Include proper controls to account for confounding variables and establish baseline measurements

What are the optimal storage conditions for recombinant FAM82A2 protein?

For optimal stability and activity of recombinant Pongo abelii FAM82A2 protein:

• Short-term storage (up to one week): Store working aliquots at 4°C

• Standard storage: Maintain at -20°C in the provided storage buffer (typically Tris-based buffer with 50% glycerol)

• Long-term storage: For extended preservation, store at -80°C

• Avoid repeated freeze-thaw cycles as they can lead to protein degradation and loss of activity

• Prepare small working aliquots to minimize the need for repeated thawing of the entire stock

These storage recommendations are critical for maintaining protein integrity and experimental reproducibility .

How can I assess the interaction between FAM82A2 and its binding partners in cellular contexts?

To study FAM82A2 protein-protein interactions in cellular contexts, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Transfect cells with tagged FAM82A2 and potential interaction partners

  • Lyse cells under non-denaturing conditions

  • Use antibodies against the tag or native FAM82A2 to precipitate protein complexes

  • Analyze precipitated proteins by western blot to detect interaction partners

• Proximity Ligation Assay (PLA):

  • Fix cells expressing FAM82A2 and potential partners

  • Use primary antibodies against both proteins

  • Apply species-specific secondary antibodies conjugated with oligonucleotides

  • If proteins are in close proximity (<40 nm), oligonucleotides can hybridize

  • Amplify signal and visualize using fluorescence microscopy

• FRET (Förster Resonance Energy Transfer):

  • Generate fusion constructs of FAM82A2 and partner proteins with appropriate fluorophores

  • Measure energy transfer between fluorophores when proteins interact

  • Quantify interaction strength and spatial arrangement

• Bioluminescence Resonance Energy Transfer (BRET):

  • Similar to FRET but uses a bioluminescent donor instead of a fluorescent one

  • Reduces background signal and allows for live-cell measurements

Each method offers distinct advantages for studying different aspects of FAM82A2 interactions with YWHAB, PTPN1, and other known partners .

What experimental approaches can be used to study FAM82A2's role in microtubule dynamics?

As a regulator of microtubule dynamics, FAM82A2 can be studied using several specialized techniques:

• Live-cell imaging with fluorescently labeled tubulin:

  • Transfect cells with fluorescent tubulin constructs

  • Compare microtubule growth, shrinkage, and catastrophe rates in cells with normal vs. altered FAM82A2 levels

  • Track individual microtubule plus-ends using markers like EB1-GFP

• In vitro reconstitution assays:

  • Purify tubulin and recombinant FAM82A2

  • Measure tubulin polymerization rates with varying FAM82A2 concentrations

  • Use total internal reflection fluorescence (TIRF) microscopy to observe individual microtubule dynamics

• CRISPR/Cas9-mediated gene editing:

  • Generate FAM82A2 knockout or mutant cell lines

  • Analyze changes in microtubule organization and dynamics

  • Rescue phenotypes with wild-type or mutant FAM82A2 constructs

• Quantitative data collection:

  Parameter	Control Cells	FAM82A2 Knockout	FAM82A2 Overexpression
  Growth rate (μm/min)	Baseline	Change (±SD)	Change (±SD)
  Shrinkage rate (μm/min)	Baseline	Change (±SD)	Change (±SD)
  Catastrophe frequency (events/min)	Baseline	Change (±SD)	Change (±SD)
  Rescue frequency (events/min)	Baseline	Change (±SD)	Change (±SD)

These approaches provide comprehensive insights into how FAM82A2 regulates microtubule behavior in cellular contexts.

What expression systems are available for producing recombinant FAM82A2?

Multiple expression systems have been validated for recombinant FAM82A2 production, each with distinct advantages:

• Mammalian expression systems:

  • HEK293 cells are commonly used for producing FAM82A2 with proper post-translational modifications

  • Ideal for functional studies requiring native protein conformation

  • Available for producing FAM82A2 from multiple species including human, mouse, rat, and Rhesus macaque

• E. coli expression systems:

  • Higher yield but may lack some post-translational modifications

  • Suitable for structural studies and some in vitro assays

  • Typically requires optimization of solubility and folding conditions

• Insect cell expression systems:

  • Baculovirus-infected insect cells offer a compromise between yield and proper folding

  • Useful for producing larger quantities while maintaining eukaryotic processing

• Available tag options:

  • His-tag: For simple purification via metal affinity chromatography

  • Fc-tag: Enhances solubility and enables protein A/G purification

  • Avi-tag: Allows for site-specific biotinylation for immobilization or detection

What purification strategies yield the highest purity and activity for recombinant FAM82A2?

For optimal purification of recombinant FAM82A2, consider this multi-step approach:

• Initial capture:

  • For His-tagged proteins: Immobilized metal affinity chromatography (IMAC)

  • For Fc-tagged proteins: Protein A/G affinity chromatography

  • Include protease inhibitors throughout purification to prevent degradation

• Intermediate purification:

  • Ion exchange chromatography based on FAM82A2's theoretical pI

  • Size exclusion chromatography to separate monomeric protein from aggregates

• Quality assessment:

  • SDS-PAGE and western blot to confirm identity and assess purity

  • Mass spectrometry to verify the intact protein mass and sequence coverage

  • Activity assays to confirm functional integrity

• Typical yield and purity metrics:

  Purification Step	Protein Recovery (%)	Purity (%)	Specific Activity
  Crude lysate	100	5-10	Baseline
  IMAC/Affinity	70-80	80-85	5-8x increase
  Ion exchange	60-70	90-95	8-10x increase
  Size exclusion	50-60	>95	10-12x increase

• Final formulation:

  • Tris-based buffer with 50% glycerol for stability

  • Aliquot and flash-freeze for long-term storage

  • Validate activity before and after storage to confirm preservation of function

What are common challenges when working with FAM82A2 and how can they be addressed?

Researchers frequently encounter these challenges when working with FAM82A2:

• Protein instability and aggregation:

  • Add reducing agents (1-5 mM DTT or β-mercaptoethanol) to buffers

  • Include 5-10% glycerol in working solutions to enhance stability

  • Filter solutions through 0.22 μm filters before use to remove aggregates

  • Consider adding stabilizing agents like trehalose or sucrose for long-term storage

• Low activity in functional assays:

  • Verify protein folding using circular dichroism or limited proteolysis

  • Test activity immediately after thawing to minimize degradation effects

  • Optimize buffer conditions (pH, salt concentration) for specific assays

  • Consider adding cofactors or interaction partners that might be required for activity

• Non-specific binding in interaction studies:

  • Increase wash stringency gradually (higher salt, mild detergents)

  • Pre-clear lysates with appropriate control beads

  • Include competing proteins (BSA, casein) to block non-specific interactions

  • Validate interactions with multiple methods (pull-down, co-IP, proximity ligation)

• Loss of microtubule regulatory activity:

  • Ensure proper post-translational modifications are present

  • Check for proper subcellular localization in cellular assays

  • Verify the integrity of key functional domains through limited proteolysis or functional mapping

How can I optimize experimental conditions for studying FAM82A2-microtubule interactions?

To maximize success in studying FAM82A2-microtubule interactions:

• Buffer optimization:

  Component	Recommended Range	Optimization Notes
  pH	6.8-7.4	Test in 0.2 pH increments
  NaCl	50-150 mM	Higher salt reduces non-specific binding
  MgCl₂	1-5 mM	Required for tubulin polymerization
  GTP	0.5-1 mM	Essential for microtubule dynamics
  Glycerol	5-10%	Stabilizes proteins, enhances microtubule formation

• Temperature considerations:

  • Pre-warm all components to 37°C for in vitro polymerization assays

  • Conduct live-cell imaging at physiological temperature (37°C)

  • Perform protein binding studies at both 4°C and room temperature to distinguish kinetic effects

• Imaging optimization:

  • Use TIRF microscopy for single-microtubule resolution

  • Consider lattice light-sheet microscopy for 3D visualization with reduced phototoxicity

  • Apply deconvolution algorithms to enhance signal-to-noise ratio in conventional microscopy

• Controls and validation:

  • Include known microtubule stabilizing (taxol) and destabilizing (nocodazole) agents as references

  • Use FAM82A2 mutants with altered binding properties as functional controls

  • Verify results with both fixed and live-cell approaches to rule out fixation artifacts

How should I analyze protein-protein interaction data involving FAM82A2?

For robust analysis of FAM82A2 interaction data:

• Quantitative co-immunoprecipitation analysis:

  • Normalize pull-down efficiency using the amount of bait protein recovered

  • Calculate enrichment ratios compared to control conditions

  • Apply statistical tests appropriate for the experimental design (t-test, ANOVA)

  • Present data as fold enrichment with error bars representing standard deviation

• Binding affinity determination:

  • For surface plasmon resonance (SPR) or microscale thermophoresis (MST) data:

    • Fit binding curves to appropriate models (1:1 binding, cooperative binding)

    • Report KD values with confidence intervals

    • Compare affinity constants across different experimental conditions

• Visualization of complex interaction networks:

  • Create interaction maps highlighting primary and secondary binding partners

  • Weight connections based on interaction strength or confidence

  • Integrate your FAM82A2 data with published interactome databases

• Sample data presentation format:

  Interaction Partner	Detection Method	Relative Binding Strength	P-value	Biological Context
  YWHAB	Co-IP	Strong (+++++)	<0.001	Cell cycle regulation
  PTPN1	PLA	Moderate (+++)	<0.01	Signaling pathway
  VAPA	FRET	Weak (+)	<0.05	Vesicular transport

What statistical approaches are appropriate for analyzing FAM82A2 functional data?

When analyzing functional data related to FAM82A2's role in microtubule dynamics:

• Time-series analysis for dynamic processes:

  • Apply regression models to quantify rates of change in microtubule length

  • Use change-point detection algorithms to identify catastrophe and rescue events

  • Implement mixed-effects models to account for cell-to-cell variability

• Comparative statistical methods:

  • One-way ANOVA with post-hoc tests for comparing multiple experimental conditions

  • Repeated measures designs for tracking changes over time within samples

  • Non-parametric alternatives (Kruskal-Wallis, Mann-Whitney) when normality assumptions are violated

• Power analysis and sample size determination:

  • Calculate minimum sample sizes needed to detect biologically meaningful effects

  • Report effect sizes (Cohen's d, partial η²) alongside p-values

  • Consider biological significance in addition to statistical significance

• Visualization strategies:

  • Box plots with individual data points for distribution comparison

  • Violin plots to visualize data density and distribution shape

  • Time-course plots with confidence intervals for dynamic processes

• Example statistical reporting format:

  Parameter	Control Mean (±SD)	FAM82A2 KO Mean (±SD)	Percent Change	Statistical Test	P-value	Effect Size
  MT Growth Rate	10.2 (±1.3) μm/min	7.6 (±1.5) μm/min	-25.5%	Student's t-test	0.002	1.85 (large)
  Catastrophe Frequency	0.058 (±0.012) events/s	0.082 (±0.017) events/s	+41.4%	Mann-Whitney U	<0.001	0.78 (medium)

This structured approach ensures robust interpretation of FAM82A2 functional data with appropriate statistical rigor.