Recombinant Mouse Uncharacterized protein C12orf70 homolog

What is Recombinant Mouse Uncharacterized protein C12orf70 homolog?

Recombinant Mouse Uncharacterized protein C12orf70 homolog is a protein expressed in Mus musculus with the Uniprot identification number Q9DA21 . It is classified as an uncharacterized protein, meaning its precise biological function remains to be fully elucidated through experimental research. The recombinant form is typically produced in E. coli expression systems to enable laboratory investigations . The full-length protein consists of 347 amino acids, though partial versions are also available for research purposes .

What are the key structural characteristics of C12orf70 homolog?

The C12orf70 homolog protein has a specific amino acid sequence that begins with MMSLQLGTAGKERQLAEKSRDLQNVSMTEGSEEVSEMDHISDRPDEKDKPSENLQTDSLY and continues through a series of defined residues . Structural analyses suggest potential functional domains, though these require further experimental validation. Researchers should note that this protein is homologous to the human C12orf70 protein, with conserved regions suggesting evolutionary significance. The amino acid sequence can be used for alignments and structural predictions to guide hypothesis generation in functional studies.

What are the recommended storage conditions for optimal stability?

For maximum stability and research reliability, the following storage conditions are recommended:

• Lyophilized form has a shelf life of approximately 12 months when stored at -20°C or -80°C

• Liquid form maintains stability for approximately 6 months at -20°C or -80°C

• Working aliquots should be stored at 4°C for no more than one week

• Repeated freeze-thaw cycles should be avoided to prevent protein degradation

Researchers should centrifuge vials briefly before opening to ensure all content is at the bottom of the tube . For long-term storage, adding glycerol to a final concentration of 50% is recommended, though this percentage can be adjusted based on specific experimental requirements .

What experimental designs are suitable for studying uncharacterized proteins like C12orf70 homolog?

When investigating uncharacterized proteins like C12orf70 homolog, researchers should consider the following experimental design approaches:

True Experimental Design:
This approach requires random assignment of subjects to control and treatment groups, with the researcher having control over the treatment variables . For protein function studies, this might involve randomly assigning cell cultures to different treatment conditions (e.g., with or without the protein of interest).

Quasi-Experimental Design:
This is appropriate when true randomization is not feasible for ethical or practical reasons . For example, when studying the protein's effects in pre-existing animal models with specific genetic backgrounds. The key characteristics of quasi-experimental design include:

Quasi-experimental designs are particularly valuable when studying protein function in complex biological systems where complete control of variables may be impossible .

How should researchers approach reconstitution of C12orf70 homolog for experimental use?

For optimal experimental results, follow these reconstitution guidelines:

• Centrifuge the vial briefly before opening to ensure the protein pellet is at the bottom

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

• For long-term storage of reconstituted protein, add glycerol to a final concentration of 5-50%

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Validate protein activity after reconstitution using appropriate functional assays

The reconstitution buffer may need optimization based on the specific experimental applications planned, particularly for functional studies that may require specific ionic conditions or pH ranges.

How can researchers verify the purity and identity of C12orf70 homolog for experimental validity?

Verification of protein purity and identity is critical for experimental reliability. The following methodological approaches are recommended:

• SDS-PAGE Analysis: The commercial recombinant protein typically shows >85% purity by SDS-PAGE . Researchers should run their own validation gels to confirm this specification.

• Western Blot Analysis: Using antibodies specific to either the protein itself or to the tag included in the recombinant construct (tag type is determined during the manufacturing process) .

• Mass Spectrometry: For precise molecular weight determination and confirmation of post-translational modifications.

• Sequence Verification: If necessary, partial sequencing can confirm the identity of the protein.

• Functional Assays: Development of activity-based assays, though challenging for uncharacterized proteins, can provide further validation.

Researchers should document all verification procedures in their experimental protocols to ensure reproducibility and reliability of subsequent findings.

What considerations are important when designing protein interaction studies with C12orf70 homolog?

When investigating potential interaction partners of this uncharacterized protein, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Design antibodies against C12orf70 homolog or utilize the tag present in the recombinant construct.

• Yeast Two-Hybrid Screening: Particularly useful for initial identification of potential binding partners.

• Proximity Labeling: Methods such as BioID or APEX can identify proteins in close proximity to C12orf70 homolog in living cells.

• Pull-down Assays: Using the recombinant protein as bait to identify binding partners from cellular lysates.

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics with candidate interaction partners.

Each approach requires careful experimental controls, including:

• Negative controls using unrelated proteins of similar size/structure

• Validation of interactions using multiple independent methods

• Consideration of tag interference with protein interactions

• Assessment of biological relevance of identified interactions

What statistical approaches are recommended for analyzing experimental data related to uncharacterized proteins?

When analyzing experimental data for uncharacterized proteins like C12orf70 homolog, researchers should employ rigorous statistical methods:

• For Comparative Studies:

  • Use t-tests for comparing two conditions (e.g., wild-type vs. knockout)

  • Employ ANOVA for multiple condition comparisons with appropriate post-hoc tests

  • Consider non-parametric alternatives when data does not meet normality assumptions

• For Correlation Studies:

  • Use Pearson's correlation for parametric data or Spearman's rank correlation for non-parametric data

  • Employ regression analysis to identify potential functional relationships

• For High-Throughput Data (e.g., from proteomics or transcriptomics):

  • Multiple testing correction methods (e.g., Benjamini-Hochberg procedure)

  • Clustering analysis to identify patterns of coregulation

  • Principal component analysis to reduce dimensionality and identify major sources of variation

• For Reproducibility:

  • Calculate confidence intervals to indicate precision of estimates

  • Report effect sizes along with p-values

  • Consider power analysis for appropriate sample size determination

How should researchers approach functional prediction for uncharacterized proteins like C12orf70 homolog?

Functional prediction for uncharacterized proteins should follow a systematic approach:

• Sequence-Based Analysis:

  • Homology detection through BLAST and HMM-based searches

  • Domain prediction using databases like Pfam, SMART, and InterPro

  • Secondary structure prediction

• Structural Analysis:

  • Homology modeling based on known structures of related proteins

  • Ab initio modeling for novel folds

  • Molecular dynamics simulations to predict dynamic behavior

• Experimental Validation:

  • Target selected predicted functions for experimental testing

  • Design experiments with appropriate positive and negative controls

  • Use multiple orthogonal approaches to validate predictions

• Integration with Omics Data:

  • Gene expression correlation analysis

  • Protein-protein interaction network analysis

  • Phenotypic data from knockout/knockdown models

It's crucial to document both confirmed and refuted functional predictions to build a comprehensive understanding of the protein's role.

What are the common challenges in studying uncharacterized proteins and how can they be addressed?

Researchers face several methodological challenges when working with uncharacterized proteins:

• Lack of Known Function:

  • Approach: Employ computational prediction tools followed by targeted experimental validation

  • Method: Use comparative genomics to identify conserved features suggesting functional importance

• Limited Reagent Availability:

  • Approach: Develop and validate custom antibodies or utilize epitope tags

  • Method: Share resources through research consortia and collaborations

• Solubility and Stability Issues:

  • Approach: Optimize buffer conditions systematically

  • Method: Consider expression of subdomains if the full-length protein proves problematic

• Determining Physiological Relevance:

  • Approach: Use gene editing techniques like CRISPR/Cas9 to study the protein in its native context

  • Method: Compare phenotypes across multiple model systems

• Reproducibility Concerns:

  • Approach: Standardize experimental protocols rigorously

  • Method: Implement blinding and randomization when possible

How can researchers resolve contradictory experimental results when studying C12orf70 homolog?

When faced with contradictory results, follow this systematic resolution approach:

• Methodological Evaluation:

  • Scrutinize experimental conditions including protein quality, concentration, and buffer composition

  • Validate all reagents used, including antibodies and expression constructs

• Statistical Reassessment:

  • Review statistical power and sample sizes

  • Consider biological vs. technical replication strategies

• Context Dependency:

  • Investigate whether contradictions arise from different cellular contexts or experimental conditions

  • Determine if post-translational modifications might explain functional differences

• Independent Validation:

  • Employ orthogonal techniques to test the same hypothesis

  • Consider collaborative validation with other laboratories

• Literature Reassessment:

  • Conduct systematic review of similar proteins for insight

  • Consider contacting authors of conflicting studies for clarification

Contradictory results often reveal important regulatory mechanisms or context-dependent functions that may be biologically significant rather than experimental artifacts.

What emerging methodologies show promise for characterizing proteins like C12orf70 homolog?

Cutting-edge methodologies that researchers should consider include:

• Cryo-Electron Microscopy:

  • Enables structural determination without crystallization

  • Particularly valuable for membrane-associated or large protein complexes

• Single-Cell Proteomics:

  • Reveals cell-type specific expression and function

  • Helps identify rare cell populations where the protein may have critical functions

• Protein Engineering Approaches:

  • Systematic mutagenesis to map functional domains

  • Creation of optogenetic or chemically-inducible variants for temporal control

• Integrative Multi-Omics Analysis:

  • Combining proteomics, transcriptomics, and metabolomics data

  • Network-based approaches to position the protein within cellular pathways

• Advanced Computational Methods:

  • Machine learning for function prediction from sequence and structure

  • Molecular dynamics simulations at extended timescales

These emerging methodologies can provide complementary insights when traditional approaches yield limited information about uncharacterized proteins.

How can researchers design longitudinal studies to track the dynamic functions of C12orf70 homolog?

Longitudinal studies require careful experimental design:

• Time Course Experimental Design:

  • Select appropriate time points based on biological processes of interest

  • Ensure statistical power at each time point with adequate replication

• Sample Collection and Preservation:

  • Standardize collection protocols to minimize technical variation

  • Consider parallel processing vs. batch processing trade-offs

• Dynamic Protein Modification Tracking:

  • Employ pulse-chase labeling to track protein turnover

  • Use phospho-specific antibodies or mass spectrometry to monitor post-translational modifications

• Statistical Analysis for Longitudinal Data:

  • Apply repeated measures ANOVA or mixed-effects models

  • Consider time series analysis methods for identifying patterns

• Systems-Level Integration:

  • Correlate protein dynamics with transcriptomic or phenotypic changes

  • Model feedback and regulatory mechanisms

Longitudinal studies are particularly valuable for understanding protein function in development, aging, or disease progression contexts.