Recombinant Macaca fascicularis Uncharacterized protein C12orf70 homolog (QtsA-14166)

What is the basic structure and sequence of Macaca fascicularis C12orf70 homolog?

The Macaca fascicularis uncharacterized protein C12orf70 homolog (QtsA-14166) consists of 293 amino acids. Its amino acid sequence is: MALTPTNLNNEMSLPMKMDCQEQELTEKNNSFFQKLNVTKSVMQDLLKEIIKVDYILDRSDDEDDISSENPQTDFLHKGMLELEAK HDQDLGKQDEQETDVDEYPQASTSLQFSKKNLLEFLUKDMLTLKGQIDKLEDRGLLDLDQGTNTEVNARNEVYELKKKVMESLE DLCKNVELLSAKLRMYQMEGENTDSHSSEETDMEEMETLLPQAPASFLVQNSPPPNTVWKCALRIFIMFYVLTVTGLLCYILFF GATFLFERVILRMLGCRTTWD LREMIEPFLNSEVEALLPS . This protein is cataloged in UniProt with the accession number Q95JR4, and it represents the full-length protein spanning residues 1-293 .

How does the C12orf70 homolog in Macaca fascicularis compare to its human counterpart?

The C12orf70 homolog in Macaca fascicularis is an uncharacterized protein that shares structural and sequence similarities with the human C12orf70 protein. As a homolog, it likely serves similar functions to its human counterpart, though specific differences in post-translational modifications or protein-protein interactions may exist. When designing cross-species experiments, researchers should consider these potential variations. Comparative studies between the macaque and human versions could provide valuable insights into evolutionary conservation of protein function, particularly in pathways that might be involved in disease mechanisms .

What are the recommended storage and handling conditions for the recombinant C12orf70 homolog protein?

The recombinant Macaca fascicularis C12orf70 homolog should be stored in Tris-based buffer with 50% glycerol, which has been optimized for this specific protein. For long-term storage, maintaining the protein at -20°C or -80°C is recommended. To preserve protein integrity, repeated freeze-thaw cycles should be avoided. Working aliquots can be stored at 4°C for up to one week . When designing experiments, researchers should consider these storage parameters to ensure protein stability throughout the experimental timeline and prevent degradation that could affect experimental outcomes.

What are the optimal experimental conditions for studying protein-protein interactions involving C12orf70 homolog?

When designing experiments to study protein-protein interactions involving the C12orf70 homolog, researchers should consider approaches similar to those used for other uncharacterized proteins. Co-immunoprecipitation (Co-IP) followed by mass spectrometry has proven effective for identifying interaction partners of novel proteins. For example, similar techniques were successfully employed to identify PAX8-interacting partners as shown in the literature .

Experimental conditions should include:

• Buffer systems that maintain physiological pH (typically 7.2-7.4)

• Inclusion of protease inhibitors to prevent degradation

• Mild detergents that solubilize membranes without disrupting protein-protein interactions

• Cross-linking approaches for transient interactions

• Controls to distinguish specific from non-specific binding

Validation of potential interaction partners should be performed using multiple techniques such as pull-down assays, surface plasmon resonance, or proximity ligation assays to establish confidence in the identified interactions.

How can researchers design effective experiments to determine the subcellular localization of C12orf70 homolog?

To determine the subcellular localization of the C12orf70 homolog, researchers should implement a multi-faceted approach:

• Immunofluorescence microscopy: Using specific antibodies against C12orf70 homolog or epitope tags if using recombinant expressions, combined with markers for different cellular compartments (nucleus, ER, Golgi, mitochondria, etc.).

• Subcellular fractionation: Physical separation of cellular components followed by Western blot analysis to detect the presence of C12orf70 in different fractions.

• Live-cell imaging: Expression of fluorescently-tagged C12orf70 to monitor its localization in real-time, particularly useful for studying dynamic processes.

• Electron microscopy: For high-resolution localization studies, especially if the protein forms specific structures.

The amino acid sequence of C12orf70 homolog suggests potential transmembrane domains (evidenced by the hydrophobic regions in the C-terminal portion: "CALRIFIMFYVLTVTGLLCYILFFGATFLFER") , which indicates possible membrane association that should be considered when designing localization experiments.

What control samples should be included when using C12orf70 homolog in transcriptome analysis studies?

When conducting transcriptome analysis involving C12orf70 homolog, comprehensive control samples are essential:

• Cell/tissue type-matched controls: Use the same cell type or tissue without C12orf70 manipulation to establish baseline expression.

• Vector controls: If overexpressing C12orf70, include empty vector transfections to account for effects of the transfection process itself.

• Wild-type protein controls: When studying mutant forms, include wild-type protein expression to differentiate mutant-specific effects.

• Time-course controls: Include samples collected at different time points to differentiate immediate vs. secondary effects of C12orf70 expression or knockdown.

• Dose-response controls: When applicable, use varying concentrations of the protein to establish dose-dependent effects.

Research on similar proteins has demonstrated that detailed experimental design accounting for different sources of variation is critical for reliable transcriptome analysis . Proper normalization and statistical analysis accounting for these variables are essential for accurate interpretation of results.

How might the C12orf70 homolog be involved in RNA processing pathways in Macaca fascicularis?

While direct evidence for C12orf70 homolog involvement in RNA processing is limited, its potential role can be investigated based on similar proteins studied in related contexts. Research on RNA binding proteins in Macaca fascicularis has identified several proteins involved in RNA processing, including zinc finger RNA-binding proteins and ATP-dependent RNA helicases .

To investigate C12orf70's potential role in RNA processing:

• RNA-protein interaction studies: Techniques such as RNA immunoprecipitation (RIP) or cross-linking immunoprecipitation (CLIP) could reveal whether C12orf70 binds specific RNA targets.

• Transcriptome analysis: Comparing RNA expression and splicing patterns in cells with normal versus altered C12orf70 levels could identify pathways affected by this protein.

• Protein domain analysis: The amino acid sequence of C12orf70 should be analyzed for known RNA-binding motifs or domains that might suggest interaction with RNA molecules.

• Functional assays: Measuring rates of transcription, RNA processing, or stability in models with modified C12orf70 expression could provide functional evidence for its role.

Similar approaches have been successfully applied to characterize other initially uncharacterized proteins in molecular biology research .

What are the methodological considerations for investigating the role of C12orf70 homolog in neurological development models using Macaca fascicularis?

Investigating C12orf70 homolog in neurological development requires careful consideration of the following methodological aspects:

• Appropriate developmental time points: Since neurological development follows specific temporal patterns, sampling should capture key developmental stages in the Macaca fascicularis model.

• Regional specificity: The central nervous system exhibits significant regional variation; therefore, precise anatomical targeting is essential when studying neurological development.

• Cell-type specificity: Consideration of neuronal versus glial expression patterns is critical, as these cell types have distinct developmental trajectories and functions.

• Comparative approach: Leveraging the anatomical and developmental similarities between Macaca fascicularis and humans can enhance translational relevance .

• Ethical considerations: Research using non-human primates requires stringent ethical oversight, particularly for neurological studies.

How can researchers address potential contradictions in experimental data when characterizing the function of uncharacterized proteins like C12orf70 homolog?

When characterizing uncharacterized proteins like C12orf70 homolog, researchers frequently encounter contradictory experimental results. To address these contradictions systematically:

• Methodological triangulation: Apply multiple distinct techniques to address the same research question. For example, if protein localization shows contradictory results, combine immunofluorescence, subcellular fractionation, and live cell imaging approaches.

• Critical evaluation of experimental conditions: Assess whether contradictions arise from differences in cell types, protein expression levels, post-translational modifications, or interaction partners that vary between experimental systems.

• Systematic variation analysis: Design experiments that systematically vary conditions (pH, salt concentration, temperature, cell cycle stage) to identify context-dependent protein behaviors.

• Computational biology approaches: Employ bioinformatic predictions and structural modeling to generate hypotheses that might explain contradictory functional data.

• Collaborative validation: Establish collaborations with independent laboratories to verify key findings using standardized protocols, reducing laboratory-specific biases.

This systematic approach to contradiction resolution has proven effective in characterizing previously uncharacterized proteins in transcriptomic studies of various model organisms .

What mass spectrometry approaches are most effective for identifying post-translational modifications of the C12orf70 homolog?

For comprehensive analysis of post-translational modifications (PTMs) in C12orf70 homolog, researchers should implement a multi-faceted mass spectrometry (MS) approach:

• Sample preparation strategies:

  • Enrichment techniques specific to the PTM of interest (phosphopeptide enrichment, ubiquitin remnant motif antibodies)

  • Multiple proteolytic enzymes beyond trypsin (chymotrypsin, Glu-C) to generate complementary peptide coverage

  • Targeted protein isolation through immunoprecipitation prior to MS analysis

• MS instrumentation and methodology:

  • High-resolution instruments (Orbitrap or Q-TOF) for accurate mass determination

  • Electron transfer dissociation (ETD) or electron capture dissociation (ECD) for labile modifications

  • Data-dependent acquisition for discovery mode and parallel reaction monitoring for targeted validation

  • Multi-stage fragmentation (MS³) for detailed structural characterization

• Data analysis pipeline:

  • Search algorithms optimized for PTM identification

  • False discovery rate control specific to PTM assignments

  • Site localization scoring for positional confidence

Similar approaches have been successfully applied to characterize PTMs on various proteins as demonstrated in studies of transcription factors and their interaction partners .

What are the key considerations for developing a specific and sensitive ELISA protocol for C12orf70 homolog detection?

Developing an optimized ELISA protocol for C12orf70 homolog requires attention to several critical parameters:

• Antibody selection and validation:

  • Validate antibody specificity using western blot and immunoprecipitation

  • Determine optimal antibody pairs for capture and detection that recognize distinct epitopes

  • Consider using monoclonal antibodies for reproducibility or polyclonal antibodies for enhanced sensitivity

• Assay optimization:

  • Establish optimal coating buffer composition and concentration

  • Determine blocking reagents that minimize background without affecting specific binding

  • Titrate antibody concentrations to achieve maximum signal-to-noise ratio

  • Optimize sample dilution to ensure measurements within the linear range of the assay

• Standard curve development:

  • Use purified recombinant Macaca fascicularis C12orf70 homolog as a reference standard

  • Prepare standards in a matrix similar to test samples

  • Include internal controls for inter-assay normalization

• Validation parameters:

  • Determine assay sensitivity (lower limit of detection and quantification)

  • Establish specificity through cross-reactivity testing

  • Assess precision through intra- and inter-assay coefficient of variation

  • Confirm accuracy through spike-recovery experiments

Each of these parameters should be systematically optimized and documented to ensure reproducible and reliable detection of C12orf70 homolog in various experimental contexts.

How can researchers effectively design cell-based assays to investigate the potential role of C12orf70 homolog in cellular signaling pathways?

To investigate C12orf70 homolog's role in cellular signaling pathways, researchers should implement the following cell-based assay design principles:

• Pathway-specific reporter systems:

  • Luciferase or fluorescent protein reporters driven by pathway-responsive elements

  • BRET/FRET-based biosensors for real-time monitoring of protein interactions

  • Phosphorylation-specific antibodies for key pathway components in high-content imaging

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated knockout models

  • Inducible expression systems (tetracycline-controlled, etc.)

  • siRNA/shRNA-mediated transient knockdown

  • Structure-guided mutagenesis of potential functional domains

• Stimulation protocols:

  • Time-course analyses to distinguish early vs. late responses

  • Dose-response studies to establish sensitivity thresholds

  • Pathway-specific activators and inhibitors to probe integration points

• Readout methodologies:

  • Multiplex analysis of pathway components (e.g., phosphoproteomics)

  • Single-cell analysis to capture population heterogeneity

  • Live-cell imaging for temporal resolution of signaling events

Previous research examining uncharacterized proteins has benefited from these approaches, particularly in establishing their roles in transcriptional regulation and signal transduction pathways .

How does the experimental approach differ when studying the C12orf70 homolog in Macaca fascicularis versus human cell systems?

When conducting comparative studies of C12orf70 homolog between Macaca fascicularis and human systems, researchers should consider several methodological differences:

• Model system selection:

  • Macaca fascicularis primary cells preserve species-specific interactions but have limited availability and greater variability

  • Immortalized Macaca cell lines offer reproducibility but may have altered signaling pathways

  • Human cell lines provide abundant material but may not recapitulate species-specific interactions

• Species-specific reagents:

  • Antibodies may exhibit different affinities between species

  • Commercial kits often optimized for human proteins may require validation in Macaca systems

  • Species-specific reference databases for omics approaches

• Experimental design adjustments:

  • Control for evolutionary differences in interacting proteins

  • Account for potential differences in post-translational modifications

  • Consider species-specific regulatory elements when studying gene expression

• Translation to human relevance:

  • Establish sequence and structural conservation between species

  • Validate key findings across species boundaries

  • Identify species-specific differences that may impact interpretation

The anatomical and physiological similarities between Macaca fascicularis and humans make this species particularly valuable for translational research , but these methodological considerations are essential for proper experimental design and interpretation.

What computational approaches are recommended for predicting the function of uncharacterized proteins like C12orf70 homolog?

For functional prediction of uncharacterized proteins like C12orf70 homolog, a multi-layered computational approach is recommended:

• Sequence-based analysis:

  • Homology detection using sensitive profile-based methods (PSI-BLAST, HHpred)

  • Domain and motif prediction using specialized databases (Pfam, PROSITE)

  • Secondary structure prediction and disorder analysis

  • Evolutionary conservation mapping to identify functional residues

• Structural bioinformatics:

  • Homology modeling based on related proteins

  • Ab initio structure prediction for novel folds

  • Molecular dynamics simulations to predict functional movements

  • Protein-protein docking to hypothesize interaction partners

• Systems biology approaches:

  • Co-expression network analysis to identify functional associations

  • Protein-protein interaction network positioning

  • Phylogenetic profiling to predict functional relationships

  • Pathway enrichment analysis of potential interaction partners

• Integration with experimental data:

  • Using available proteomics data to validate predictions

  • Incorporating transcriptomic data to understand expression patterns

  • Leveraging ChIP-seq or similar data to predict regulatory roles

These computational approaches have been successfully applied to characterize previously uncharacterized proteins, as demonstrated in transcriptomic studies and protein interaction analyses .

What are the current limitations in our understanding of C12orf70 homolog and what research directions show the most promise?

Current understanding of the C12orf70 homolog in Macaca fascicularis remains limited, with significant knowledge gaps regarding its physiological function, interaction partners, and involvement in cellular pathways. The protein remains classified as "uncharacterized," highlighting the need for comprehensive functional characterization .

Key limitations and promising research directions include:

• Functional annotation: Despite available sequence information, functional roles remain largely unknown. Systematic approaches combining computational prediction with experimental validation show promise for functional elucidation.

• Interactome characterization: Identifying protein-protein interactions will be crucial for placing C12orf70 within cellular networks. Techniques similar to those used for PAX8-interacting partner identification could prove valuable .

• Evolutionary significance: Comparative studies between human and macaque C12orf70 could reveal evolutionary conservation patterns that suggest functional importance.

• Tissue-specific roles: Investigating expression patterns across different tissues in Macaca fascicularis might reveal specialized functions, particularly in neurological contexts given the value of this model for neurological research .

• Disease relevance: Exploring potential associations with disease states, similar to approaches used in studies of fragile X mental retardation and premutation carriers , could provide insights into pathological significance.