Recombinant Bovine Regulator of microtubule dynamics protein 2 (FAM82A1)

What is the cellular localization pattern of FAM82A1?

FAM82A1 displays distinct localization patterns depending on the cell cycle phase:

• During interphase: Localizes primarily to the cytoplasm, with specific distribution in the microtubule lattice and perinuclear region

• During cell division: Concentrates at spindle microtubules and spindle poles

This dynamic localization pattern suggests a critical role in microtubule organization throughout the cell cycle. Visualization of these patterns typically requires immunofluorescence techniques using specific antibodies against FAM82A1, such as those described in search result .

What expression systems are commonly used for recombinant FAM82A1 production?

Several expression systems have been documented for the production of recombinant FAM82A1:

The choice of expression system depends on the research application. E. coli systems are commonly used for high yield and cost-effectiveness, while yeast and mammalian systems may provide more appropriate post-translational modifications .

Experimental Methodologies

For optimal stability and functionality of recombinant FAM82A1:

• Store lyophilized protein at -20°C to -80°C upon receipt

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

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

• Add glycerol to a final concentration of 5-50% for long-term storage

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

• Typical storage buffer: Tris/PBS-based buffer with 6% Trehalose, pH 8.0

Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided.

How can researchers utilize FAM82A1 in microtubule dynamics studies?

To study FAM82A1's role in microtubule dynamics, researchers can employ several approaches:

• In vitro microtubule assembly/disassembly assays:

  • Purified recombinant FAM82A1 can be added to tubulin polymerization reactions

  • Effects on microtubule nucleation, growth rate, and stability can be monitored using fluorescently labeled tubulin

• Live-cell imaging:

  • Express fluorescently-tagged FAM82A1 in cells

  • Monitor its co-localization with microtubules during different cell cycle phases

  • Track its association with dynamic microtubule plus-ends

• Knockdown/knockout studies:

  • Use siRNA or CRISPR-Cas9 to deplete FAM82A1

  • Analyze changes in microtubule array organization and dynamics

  • Assess effects on spindle formation and cell division

• Domain-specific functional analysis:

  • Generate truncated or mutated versions of FAM82A1

  • Test their ability to bind microtubules and affect their dynamics

  • Map functional domains critical for microtubule regulation

What is known about genetic variants in FAM82A1 and their potential disease associations?

Genome-wide association studies have identified FAM82A1 variants in several contexts:

What methodological approaches can be used to study FAM82A1 interactions with other proteins?

To characterize the FAM82A1 interactome, researchers can employ:

• Co-immunoprecipitation (Co-IP):

  • Use anti-FAM82A1 antibodies to pull down protein complexes from cell lysates

  • Identify interacting partners by mass spectrometry

  • Verify specific interactions with candidate proteins by western blotting

• Yeast two-hybrid screening:

  • Use FAM82A1 as bait to screen cDNA libraries for interacting proteins

  • Validate positive interactions through secondary assays

• Proximity labeling techniques:

  • Express FAM82A1 fused to BioID or APEX2 enzymes

  • Identify proteins in close proximity through biotinylation

  • Analyze biotinylated proteins by mass spectrometry

• Fluorescence resonance energy transfer (FRET):

  • Tag FAM82A1 and potential partners with compatible fluorophores

  • Measure energy transfer as indication of protein-protein interactions

  • Apply in live cells to capture dynamic interactions

These approaches would help establish FAM82A1's role within the wider microtubule regulatory network.

How can researchers analyze expression patterns of FAM82A1 across different tissues and conditions?

To comprehensively analyze FAM82A1 expression:

• RNA-seq analysis:

  • Analyze transcriptome data from diverse tissues and cell types

  • Compare expression levels under different conditions

  • Identify tissue-specific isoforms

• qRT-PCR:

  • Design primers specific to FAM82A1 (avoiding pseudogenes)

  • Quantify expression relative to housekeeping genes

  • Profile expression changes in response to perturbations

• Protein expression analysis:

  • Western blotting with validated antibodies (like ABIN7253428)

  • Immunohistochemistry on tissue sections

  • Flow cytometry for quantitative single-cell analysis

• Reporter assays:

  • Clone the FAM82A1 promoter region upstream of reporter genes

  • Study transcriptional regulation in different cell types

  • Identify regulatory elements controlling expression

Based on available data, FAM82A1 shows relatively high expression in neural tissues, suggesting important functions in the nervous system .

What bioinformatic approaches are useful for comparative analysis of FAM82A1 orthologs?

For comparative analysis of FAM82A1 orthologs across species:

• Sequence alignment and phylogenetic analysis:

  • Multiple sequence alignment of FAM82A1 proteins from different species

  • Construction of phylogenetic trees to infer evolutionary relationships

  • Identification of conserved domains and species-specific variations

• Protein structure prediction:

  • Use homology modeling to predict 3D structures

  • Compare structural features across species

  • Identify conserved structural elements

• Synteny analysis:

  • Examine conservation of genomic context around FAM82A1

  • Identify conserved neighboring genes

  • Infer evolutionary history of genomic rearrangements

• Functional domain analysis:

  • Identify conserved functional motifs across species

  • Compare coiled-coil domains and other structural features

  • Predict species-specific functional adaptations

Available data indicates high conservation of FAM82A1 across mammalian species, with orthologs identified in human (RMDN2), mouse (Fam82a1), and other organisms .

What are the current gaps in understanding FAM82A1 function and how might they be addressed?

Several knowledge gaps exist in FAM82A1 research:

• Precise molecular mechanism:

  • How FAM82A1 interacts with and regulates microtubules remains incompletely characterized

  • Advanced structural studies (X-ray crystallography, cryo-EM) of FAM82A1-microtubule complexes would provide mechanistic insights

• Regulatory pathways:

  • The upstream regulators and downstream effectors of FAM82A1 are largely unknown

  • Phosphoproteomic analysis could identify regulatory modifications

  • CRISPR screens might reveal genetic interactions

• Physiological significance:

  • The phenotypic consequences of FAM82A1 deletion in animal models are not well documented

  • Development of conditional knockout models would help characterize tissue-specific functions

• Disease relevance:

  • Beyond tentative associations with asthma, the role of FAM82A1 in human diseases remains unexplored

  • Patient-derived samples could be analyzed for alterations in FAM82A1 expression or function

Addressing these gaps will require interdisciplinary approaches combining structural biology, cell biology, genetics, and clinical research.

What emerging technologies might advance the study of FAM82A1 function?

Emerging technologies with potential to advance FAM82A1 research include:

• CRISPR-based technologies:

  • Base editing for precise modification of FAM82A1 at endogenous loci

  • CRISPRi/CRISPRa for temporal control of expression

  • CRISPR screens to identify genetic interactions

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize FAM82A1-microtubule interactions at nanoscale resolution

  • Lattice light-sheet microscopy for long-term live imaging with minimal phototoxicity

  • Correlative light and electron microscopy (CLEM) to bridge dynamic imaging with ultrastructural detail

• Single-cell multi-omics:

  • Single-cell transcriptomics to capture cell-type-specific expression patterns

  • Single-cell proteomics to analyze protein-level variation

  • Integration of multi-omic data for comprehensive understanding

• Organoid and tissue engineering approaches:

  • Study FAM82A1 function in 3D tissue contexts

  • Examine cell-type specific roles in complex tissues

  • Test effects of FAM82A1 manipulation in physiologically relevant models

These technologies could provide unprecedented insights into FAM82A1 biology at molecular, cellular, and organismal levels.

What are common challenges when working with recombinant FAM82A1 and how can they be addressed?

Researchers frequently encounter these challenges when working with recombinant FAM82A1:

• Protein solubility issues:

  • Problem: Recombinant FAM82A1 may form inclusion bodies in E. coli

  • Solution: Optimize expression conditions (lower temperature, reduced IPTG)

  • Alternative: Express as fusion protein with solubility tags (MBP, SUMO)

• Protein stability concerns:

  • Problem: Protein degradation during purification or storage

  • Solution: Include protease inhibitors during purification

  • Alternative: Add stabilizing agents like glycerol (5-50%) to storage buffer

• Antibody specificity:

  • Problem: Cross-reactivity with related proteins

  • Solution: Validate antibodies using knockout/knockdown controls

  • Alternative: Use epitope-tagged recombinant proteins when possible

• Functional assay optimization:

  • Problem: Variable activity in functional assays

  • Solution: Ensure proper protein folding through circular dichroism analysis

  • Alternative: Include positive controls with known activity

Addressing these challenges requires careful optimization of experimental conditions and appropriate controls.

How can researchers validate the specificity of FAM82A1 antibodies for experimental applications?

To ensure antibody specificity for FAM82A1:

• Western blot validation:

  • Run lysates from multiple species to confirm cross-reactivity claims

  • Include both positive controls (FAM82A1-overexpressing cells) and negative controls (FAM82A1-knockout cells)

  • Validate that the observed band matches the expected molecular weight (~47 kDa for human FAM82A1)

• Immunoprecipitation tests:

  • Perform IP followed by mass spectrometry to confirm target identity

  • Conduct reciprocal IPs with different antibodies targeting distinct epitopes

• Immunofluorescence validation:

  • Compare staining patterns with published localization data

  • Perform siRNA knockdown to confirm reduction in signal

  • Co-stain with microtubule markers to verify proper localization during cell division

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Confirm specific signal disappearance in presence of competing peptide

Multiple commercial antibodies are available for FAM82A1 detection, with varying specifications for species reactivity and applications .