Recombinant Mouse 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 more formally known as Regulator of microtubule dynamics protein 3 (RMDN3). The protein has multiple synonyms including FAM82C, PTPIP51 (Protein tyrosine phosphatase-interacting protein 51), RMD3, RMD-3, Cerebral protein 10, and TCPTP-interacting protein 51 (TCPTP: T-cell protein tyrosine phosphatase) . These various nomenclatures reflect its discovery in different contexts and highlight the protein's multifunctional nature in cellular processes.

What is the molecular weight and structure of mouse Fam82a2?

Mouse Fam82a2 has a molecular weight of approximately 60 kDa as determined by SDS-PAGE analysis . The protein contains specific binding domains that facilitate its interactions with various cellular components. When examining recombinant versions of the protein, it's important to account for additional mass contributed by fusion tags such as His, Avi, or Fc tags, which are commonly used in research applications . The full-length protein contains specific domains that mediate its interactions with microtubules and various binding partners.

What are the known functional domains of Fam82a2?

Fam82a2 contains specific domains that facilitate its role in microtubule dynamics regulation and cellular calcium homeostasis. The protein's structure enables its participation in protein-protein interactions with various binding partners . The functional domains are conserved across species, particularly between human and mouse versions, allowing for translational research between model systems. Each domain contributes to specific aspects of the protein's functionality, including its ability to interact with the cytoskeleton and influence cellular processes like differentiation and apoptosis.

What are the primary cellular functions of Fam82a2?

Fam82a2 is primarily involved in the regulation of cellular calcium homeostasis, which is crucial for numerous cellular processes. Additionally, it may participate in the differentiation and apoptosis of keratinocytes, suggesting a role in cellular development and programmed cell death . Research has shown that overexpression of Fam82a2 induces apoptosis, indicating its potential involvement in cell death pathways . The protein's ability to regulate microtubule dynamics also suggests a role in cellular structure maintenance and intracellular transport processes, which are essential for proper cell function and division.

How does Fam82a2 interact with other proteins in cellular pathways?

Fam82a2 demonstrates protein binding capabilities and interacts with several key proteins including YWHAB, YWHAG, PTPN1, VAPA, Rmdn3, PSEN1, and EGFL8 . These interactions have been detected through various methods such as yeast two-hybrid assays, co-immunoprecipitation, and pull-down experiments. The interaction with PTPN1 (Protein tyrosine phosphatase non-receptor type 1) is particularly noteworthy as suggested by one of its synonyms, PTPIP51 (Protein tyrosine phosphatase-interacting protein 51) . These protein interactions suggest that Fam82a2 may function within complex protein networks, potentially influencing multiple cellular pathways simultaneously.

What role does Fam82a2 play in apoptosis pathways?

Research indicates that overexpression of Fam82a2 induces apoptosis, suggesting it may function as a pro-apoptotic factor . The protein likely influences apoptotic pathways through its involvement in calcium homeostasis regulation, as calcium signaling is a known regulator of apoptotic processes. Additionally, Fam82a2's interactions with proteins like PSEN1 (Presenilin-1), which is involved in apoptosis regulation, further support its role in programmed cell death pathways . Understanding these mechanisms could provide insights into cellular death processes in both normal development and pathological conditions.

What are the optimal conditions for working with recombinant mouse Fam82a2 protein?

When working with recombinant mouse Fam82a2, researchers should store the protein at -20°C and avoid repeated freeze-thaw cycles to maintain stability and activity . Typical recombinant preparations are supplied in phosphate-buffered saline with additives such as glycerol (often 50%) and sodium azide (0.02%) to enhance stability . For experimental use, the protein is typically maintained in Tris-based buffer with 50% glycerol . When designing experiments, researchers should consider the protein's purity (typically >90% as determined by SDS-PAGE) and validate its activity using appropriate functional assays before proceeding with complex experiments.

What expression systems are most effective for producing recombinant mouse Fam82a2?

Recombinant mouse Fam82a2 can be successfully expressed in various systems, with mammalian cell expression being particularly effective for maintaining proper folding and post-translational modifications . HEK293 cells are commonly used for expression of mammalian proteins like Fam82a2, especially when proper folding and post-translational modifications are crucial for protein function . E. coli systems may also be used for expressing portions of the protein or when post-translational modifications are not essential for the experimental purpose . The choice of expression system should align with the specific research questions being addressed and the downstream applications planned.

The effectiveness of different expression systems for mouse Fam82a2 is summarized in the following table:

What antibody-based detection methods are most reliable for studying Fam82a2?

For detecting Fam82a2 in experimental samples, polyclonal antibodies that recognize endogenous levels of the total protein are available and validated for Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) applications . These antibodies have demonstrated reactivity across multiple species including human, mouse, and rat, making them versatile tools for comparative studies . When selecting an antibody, researchers should consider specificity, sensitivity, and validated applications. For Western blot applications, antibodies can detect Fam82a2 at approximately 60 kDa, and validation data using various cell lines is typically available from manufacturers .

How can knockout or knockdown models of Fam82a2 be developed and validated?

To develop effective knockout or knockdown models of Fam82a2, researchers can employ CRISPR-Cas9 genome editing for permanent gene disruption or RNA interference (RNAi) techniques for transient knockdown. Validation of these models should include genomic verification (for knockouts), RNA expression analysis via RT-qPCR, and protein expression assessment using validated antibodies against Fam82a2 . Functional validation should examine downstream effects on calcium homeostasis and apoptotic pathways, as these are known functions of the protein . Additionally, monitoring interactions with known binding partners such as YWHAB, YWHAG, or PTPN1 can provide further confirmation of successful manipulation of Fam82a2 expression .

What are the considerations for studying Fam82a2's role in calcium homeostasis?

When investigating Fam82a2's role in calcium homeostasis, researchers should employ calcium imaging techniques such as fluorescent calcium indicators (e.g., Fura-2, Fluo-4) or genetically encoded calcium indicators (GECIs) to monitor dynamic changes in intracellular calcium levels. Experimental designs should include both basal conditions and calcium challenge assays to assess how Fam82a2 manipulation affects calcium handling under various physiological states . Comparison between wildtype cells and those with altered Fam82a2 expression (overexpression, knockdown, or knockout) can reveal specific aspects of calcium regulation influenced by the protein. Additionally, co-immunoprecipitation experiments can identify calcium-dependent protein interactions that may mediate Fam82a2's effects on calcium homeostasis.

How does mouse Fam82a2 compare functionally to its human ortholog?

The mouse and human versions of Fam82a2/RMDN3 share significant sequence homology and functional conservation, making mouse models valuable for studying processes relevant to human biology. Both proteins are involved in cellular calcium homeostasis regulation and potentially in apoptotic pathways . When designing translational research, it's important to validate key findings in both species, as there may be subtle differences in protein interactions or regulatory mechanisms. Structural analysis reveals conservation of key functional domains between species, suggesting evolutionary preservation of the protein's core functions . This conservation supports the use of mouse models for studying Fam82a2-related processes with potential human health implications.

How can researchers differentiate between direct and indirect effects of Fam82a2 manipulation?

To distinguish between direct and indirect effects of Fam82a2 manipulation, researchers should employ multiple complementary approaches. Acute vs. chronic manipulation studies can help separate immediate (likely direct) effects from adaptive (potentially indirect) responses. Rescue experiments, where wildtype Fam82a2 is reintroduced into knockout models, can confirm specificity of observed phenotypes . Protein interaction studies, including proximity ligation assays or FRET-based approaches, can identify direct binding partners that mediate Fam82a2's functions. Additionally, time-course experiments following Fam82a2 manipulation can reveal the temporal sequence of cellular responses, helping to establish causality and distinguish primary from secondary effects.

What are the appropriate controls for Fam82a2 overexpression studies?

When conducting Fam82a2 overexpression studies, researchers should include several critical controls. First, empty vector controls are essential to account for effects of the transfection/transduction process itself. Second, overexpression of an unrelated protein of similar size can control for non-specific effects of protein overproduction . For tagged Fam82a2 constructs, parallel experiments with differently tagged versions (e.g., N-terminal vs. C-terminal tags) can ensure tag position doesn't interfere with protein function . Expression level monitoring is crucial, as physiologically relevant overexpression may produce different effects than extreme overexpression. Finally, dose-response studies can help establish thresholds for Fam82a2's effects on processes like apoptosis induction.

How should contradictory findings regarding Fam82a2 function be reconciled?

When faced with contradictory findings regarding Fam82a2 function, researchers should systematically examine several key variables. Cell type-specific effects may explain discrepancies, as Fam82a2's function could vary between different cellular contexts. Different experimental techniques may have varying sensitivities or limitations that influence results . Protein expression levels should be carefully considered, as concentration-dependent effects are common in protein biology. Interaction with different binding partners in various experimental systems might alter Fam82a2's functionality . Researchers should also examine differences in recombinant protein preparation (tags, expression systems) that might affect activity . Ultimately, integrating findings from multiple approaches and model systems is essential for developing a comprehensive understanding of Fam82a2's complex biology.

What are promising research applications for recombinant mouse Fam82a2?

Recombinant mouse Fam82a2 offers several promising research applications. It can serve as a valuable tool for studying protein-protein interactions, particularly with its known binding partners like YWHAB, YWHAG, PTPN1, and others . The protein can be used to develop and validate antibodies for detecting endogenous Fam82a2 in various experimental contexts . Structure-function studies using recombinant protein fragments can map domains responsible for specific interactions or functions. Additionally, the recombinant protein can serve as a standard in quantitative assays measuring endogenous Fam82a2 levels. Given its role in apoptosis, recombinant Fam82a2 could also be used to study mechanisms of programmed cell death in various cell types .

How might Fam82a2 research contribute to understanding disease mechanisms?

Research on Fam82a2 has potential implications for understanding several disease mechanisms. Given its role in calcium homeostasis regulation, Fam82a2 may be relevant to disorders involving calcium dysregulation, including neurodegenerative diseases and cardiomyopathies . Its involvement in apoptotic pathways suggests potential relevance to cancer biology, where apoptotic mechanisms are frequently disrupted . The protein's interaction with PSEN1 (Presenilin-1) points to possible connections with Alzheimer's disease pathways, as PSEN1 is associated with familial Alzheimer's disease . Additionally, understanding Fam82a2's role in cellular differentiation could provide insights into developmental disorders or regenerative medicine applications. Future research should explore these potential disease connections through targeted studies in relevant model systems.

What emerging technologies might advance Fam82a2 research?

Several emerging technologies show promise for advancing Fam82a2 research. CRISPR-based technologies beyond gene knockout, such as CRISPRa/CRISPRi for endogenous gene regulation, could provide more nuanced manipulation of Fam82a2 expression. Proximity labeling approaches like BioID or APEX could map the complete Fam82a2 interactome in various cellular contexts . Advanced imaging techniques including super-resolution microscopy could better define Fam82a2's subcellular localization and dynamics. Single-cell analyses might reveal cell-type specific functions or expression patterns currently obscured in bulk analyses. Computational approaches integrating multiple -omics datasets could place Fam82a2 within broader regulatory networks. Finally, cryo-electron microscopy could provide detailed structural information about Fam82a2 and its complexes with interaction partners, informing structure-based functional studies.

How can specificity issues with Fam82a2 antibodies be addressed?

When encountering specificity issues with Fam82a2 antibodies, researchers should implement several validation strategies. Comparing multiple antibodies raised against different epitopes can confirm true positive signals . Antibody validation in knockout/knockdown models provides the gold standard for specificity confirmation. Pre-absorption controls, where the antibody is pre-incubated with recombinant Fam82a2 before use, can identify non-specific binding. Western blot analysis should verify a single band of appropriate molecular weight (approximately 60 kDa) . For immunostaining applications, peptide competition assays and comparison with mRNA expression patterns (e.g., through in situ hybridization) can provide additional validation. Finally, testing antibodies across multiple applications (WB, IHC, IF) can build confidence in their specificity and utility for particular experimental approaches .

What strategies can overcome challenges in detecting endogenous Fam82a2 expression?

Detection of endogenous Fam82a2 can be challenging due to potentially low expression levels in some cell types. To overcome this, researchers can employ signal amplification techniques such as tyramide signal amplification for immunostaining or highly sensitive chemiluminescent substrates for Western blotting . Enrichment of samples through subcellular fractionation or immunoprecipitation before detection can concentrate the protein of interest. RT-qPCR provides a complementary approach for detecting Fam82a2 at the mRNA level, which can be particularly useful when protein detection is difficult. When using antibodies, selecting those validated for endogenous protein detection and optimizing protocols for specific sample types is crucial . Finally, comparing expression across multiple cell lines known to express different levels of Fam82a2 can help establish the sensitivity threshold of detection methods.

How can researchers ensure activity of recombinant Fam82a2 in functional assays?

To ensure recombinant Fam82a2 maintains its activity in functional assays, researchers should implement several quality control measures. Fresh preparation or minimal freeze-thaw cycles help maintain protein integrity . Validation through binding assays with known interaction partners like YWHAB, YWHAG, or PTPN1 can confirm functional capacity . For studies of calcium homeostasis effects, preliminary dose-response experiments can identify optimal concentrations for biological activity . Proper storage conditions (-20°C, in buffer with stabilizers like glycerol) are essential for maintaining protein stability between experiments . Different tags may affect activity, so comparing results with differently tagged versions or tag-cleaved protein can identify potential artifacts. Finally, parallel experiments with endogenously expressed Fam82a2 can serve as a reference point for normal functional activity levels.