Recombinant Rat Regulator of microtubule dynamics protein 3 (Fam82a2)

What is Fam82a2 and what are its known synonyms in scientific literature?

Fam82a2 is officially known as Regulator of Microtubule Dynamics Protein 3 (RMDN3). The protein is also referred to by several synonyms in scientific literature including FAM82C, ptpip51, RMD-3, and RMD3 . These alternative designations reflect its evolutionary history and functional characterization across different research groups. When conducting literature searches, researchers should include all of these terms to ensure comprehensive coverage of relevant publications.

What are the optimal storage conditions for recombinant Fam82a2 protein to maintain its stability?

Recombinant Fam82a2 protein should be stored at -20°C for regular use, or at -80°C for extended storage periods . The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which helps maintain stability during freeze-thaw cycles. To minimize protein degradation:

• Avoid repeated freeze-thaw cycles, which can significantly reduce protein activity

• Prepare small working aliquots upon first thaw

• Store working aliquots at 4°C for up to one week

• Ensure complete thawing and gentle mixing before use

For maximum recovery after freezing, centrifuge the original vial after thawing and prior to removing the cap to collect all the material at the bottom of the tube .

How should researchers design knockdown experiments to study Fam82a2 function?

When designing siRNA-mediated knockdown experiments for Fam82a2 (RMDN3), researchers should consider:

• Use of validated siRNA sequences specifically targeting rat Fam82a2 such as:

  • 5′-CCUUUAAUGUCAUACCUUA-3′

  • 5′-GCUUUAGCUUCAAGGAACA-3′

  • 5′-GCUACAGCCUUGUUUGAAA-3′

  • 5′-CUCUGGACCUUGAUAUGGA-3′

• Include appropriate controls:

  • Non-targeting control siRNAs to account for non-specific effects

  • Positive controls targeting housekeeping genes to confirm transfection efficiency

  • Vehicle-only controls to establish baseline expression

• Validation of knockdown efficiency:

  • Western blot analysis using anti-PTPIP51 antibodies

  • qRT-PCR to measure mRNA levels

  • Immunofluorescence to assess protein reduction at cellular level

Given Fam82a2's role in endoplasmic reticulum-mitochondria tethering, researchers should monitor potential changes in subcellular organelle distribution and interactions following knockdown .

How is Fam82a2/RMDN3 involved in endoplasmic reticulum-mitochondria tethering?

Fam82a2 (PTPIP51) has been identified as a key component in endoplasmic reticulum (ER)-mitochondria tethering complexes. The protein interacts with vesicle-associated membrane protein-associated protein B (VAPB) to form a tethering complex between these two organelles . This interaction:

• Facilitates calcium exchange between ER and mitochondria

• Regulates mitochondrial function and dynamics

• Influences lipid metabolism and transfer between organelles

• May play roles in apoptotic signaling pathways

Experimental approaches to study this function include co-immunoprecipitation assays to detect VAPB-PTPIP51 interactions, proximity ligation assays to visualize interaction in situ, and subcellular fractionation followed by immunoblotting to detect enrichment in ER-mitochondria contact sites .

What are the primary research applications for recombinant Fam82a2 protein?

Recombinant Fam82a2 protein serves multiple research applications:

• As a standard in quantitative assays such as ELISA and Western blotting

• For antibody production and validation

• In protein-protein interaction studies, particularly examining:

  • Binding partners in microtubule regulation pathways

  • Interactions with VAPB and other ER-mitochondria tethering proteins

• For structural studies including X-ray crystallography or cryo-EM

• As a positive control in experiments examining cellular localization and functional assays

When using recombinant Fam82a2 for these applications, researchers should note that the tag type may vary depending on the production process and should verify compatibility with their specific experimental system .

What techniques are recommended for detecting Fam82a2 expression in rat tissue samples?

Several complementary techniques are recommended for detecting Fam82a2 expression in rat tissues:

For synaptoneurosome preparations from rat brain, researchers have successfully used biochemical fractionation followed by immunoblotting with antibodies against PTPIP51 to study synaptic localization of this protein .

How can researchers verify the purity and functionality of recombinant Fam82a2 protein?

To verify recombinant Fam82a2 protein purity and functionality, researchers should employ a multi-faceted approach:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (expect >90% purity)

  • Western blot with anti-PTPIP51/Fam82a2 antibodies

  • Mass spectrometry for precise molecular weight determination

• Functionality assessment:

  • Binding assays with known interaction partners (e.g., VAPB)

  • Microtubule co-sedimentation assays to confirm interaction with tubulin

  • Circular dichroism to verify proper protein folding

  • Limited proteolysis to assess structural integrity

• Activity verification:

  • In vitro microtubule dynamics assays

  • Mitochondrial tethering assays in reconstituted systems

Researchers should consider that recombinant proteins may require specific buffers and conditions to maintain their native conformation and activity.

How should researchers approach contradictory data when studying Fam82a2 function?

When facing contradictory data in Fam82a2 research, follow these methodological steps:

• Thoroughly examine the data to identify specific discrepancies and patterns that contradict initial hypotheses .

• Evaluate experimental design factors that might contribute to unexpected results:

  • Cell or tissue-specific differences in Fam82a2 expression or function

  • Variations in protein tag types affecting protein interactions

  • Buffer composition differences affecting protein stability

  • Influence of post-translational modifications

• Consider alternative explanations for contradictory findings:

  • Context-dependent protein functions in different cellular compartments

  • Compensatory mechanisms in knockout/knockdown models

  • Existence of protein isoforms with distinct functions

  • Technical limitations in detection methods

• Implement validation experiments:

  • Use multiple approaches to measure the same parameter

  • Test in different model systems

  • Employ both gain-of-function and loss-of-function approaches

  • Collaborate with other labs for independent verification

Remember that unexpected results often lead to new discoveries about protein function, particularly for multifunctional proteins like Fam82a2 that participate in complex cellular processes .

What are common technical challenges when working with recombinant Fam82a2 and how can they be overcome?

Common technical challenges and solutions when working with recombinant Fam82a2 include:

• Protein aggregation:

  • Add stabilizing agents like glycerol (5-10%) to storage buffer

  • Optimize buffer conditions (pH, salt concentration)

  • Consider adding reducing agents if protein contains disulfide bonds

  • Store in small aliquots to avoid repeated freeze-thaw cycles

• Low antibody sensitivity:

  • Test multiple commercial antibodies

  • Consider epitope accessibility in different applications

  • Use recombinant Fam82a2 as a positive control

  • Optimize blocking conditions to reduce background

• Variable knockdown efficiency:

  • Test multiple siRNA sequences targeting different regions

  • Optimize transfection conditions for specific cell types

  • Consider stable knockdown with shRNA for long-term studies

  • Validate knockdown at both mRNA and protein levels

• Inconsistent immunolocalization:

  • Use multiple fixation protocols (PFA vs. methanol)

  • Verify antibody specificity with knockdown controls

  • Employ organelle markers for colocalization studies

  • Consider live-cell imaging with fluorescently tagged Fam82a2

How can researchers effectively study the role of Fam82a2 in ER-mitochondria communication?

Advanced approaches to study Fam82a2's role in ER-mitochondria communication include:

• Proximity-based labeling techniques:

  • BioID or APEX2 fusion proteins to identify proteins in close proximity to Fam82a2

  • Split-BioID system to detect dynamic interactions at ER-mitochondria contact sites

• Advanced microscopy approaches:

  • Super-resolution microscopy (STORM/PALM) to visualize nanoscale organization

  • Live-cell FRET sensors to measure protein-protein interactions in real-time

  • Correlative light and electron microscopy to precisely locate Fam82a2 at contact sites

• Reconstitution systems:

  • Liposome-based reconstitution of ER-mitochondria tethers

  • Optical tweezers to measure tethering forces

  • In vitro calcium transfer assays between reconstituted organelles

• Genetically encoded sensors:

  • Targeted calcium indicators to measure local Ca²⁺ transfer

  • FRET-based tension sensors to detect mechanical forces at contact sites

  • Split-GFP complementation to visualize contact formation

These advanced techniques can reveal mechanistic insights into how Fam82a2 regulates inter-organelle communication and how its dysfunction might contribute to pathological states .

What are emerging approaches for studying the interactome of Fam82a2 in different neural cell types?

Cutting-edge approaches for characterizing the Fam82a2 interactome in neural contexts include:

• Cell type-specific proximity labeling:

  • AAV-mediated expression of Fam82a2-BioID/APEX2 in specific neural populations

  • Conditional expression systems for temporal control of labeling

  • Single-cell interactome analysis using multiplexed approaches

• Spatial transcriptomics and proteomics:

  • MERFISH combined with proximity labeling to map regional interactome differences

  • Laser capture microdissection followed by mass spectrometry

  • Spatial proteomics to identify subcellular compartment-specific interactions

• Functional interaction mapping:

  • CRISPR screens to identify genetic modifiers of Fam82a2 phenotypes

  • Chemogenetic approaches to acutely modulate Fam82a2 function

  • Optogenetic control of protein-protein interactions to determine functional consequences

• Computational approaches:

  • Molecular dynamics simulations of Fam82a2 interactions

  • Machine learning algorithms to predict context-dependent interactions

  • Network analysis to identify hub proteins connected to Fam82a2

These advanced techniques can reveal cell type-specific functions of Fam82a2 in the nervous system and potential relationships to neurological disorders.