Recombinant Mouse Uncharacterized membrane protein C1orf95 homolog

What is the Recombinant Mouse Uncharacterized membrane protein C1orf95 homolog?

Recombinant Mouse Uncharacterized membrane protein C1orf95 homolog (UniProt ID: Q0VBF8) is a full-length protein consisting of 141 amino acids. It is also known as Protein stum homolog or STUM. The protein is typically expressed in E. coli as a recombinant product with an N-terminal His tag for purification purposes. This protein represents the mouse homolog of the human C1orf95 gene product, which remains functionally uncharacterized despite being identified in genomic analyses .

What structural characteristics define this membrane protein?

Based on sequence analysis, the C1orf95 homolog protein contains hydrophobic regions consistent with membrane-spanning domains. The protein sequence demonstrates characteristics of a transmembrane protein with hydrophobic segments (particularly in regions containing sequences like "FVPGLGTFVSAFTVLCGARTDLPDRH" and "IAAALIQVLTAIVMVGWIMSIFWG"), which likely anchor it within biological membranes. The protein appears to have both hydrophilic and hydrophobic domains, suggesting it may traverse the membrane with portions extending into both intracellular and extracellular spaces .

What expression systems are optimal for producing this recombinant protein?

The available data indicates that E. coli expression systems have been successfully employed to produce recombinant Mouse C1orf95 homolog protein. When expressing this protein, researchers typically use bacterial expression vectors that incorporate an N-terminal His tag to facilitate purification. Alternative expression systems such as mammalian or insect cell lines might provide better post-translational modifications if required for functional studies, though empirical validation would be necessary. The current commercially available preparations utilize E. coli expression systems optimized for membrane protein production .

What purification strategies are effective for this membrane protein?

Given the presence of the His tag in the recombinant form, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-based resins represents the primary purification approach. For membrane proteins like C1orf95 homolog, it's essential to incorporate appropriate detergents during extraction and purification to maintain protein solubility and native conformation. A typical purification protocol would involve:

• Cell lysis under native conditions with appropriate detergents

• IMAC purification using His-tag affinity

• Optional secondary purification via size exclusion chromatography

• Final formulation in a stabilizing buffer with possible detergent inclusion

How should researchers design experiments to investigate the function of this uncharacterized protein?

To investigate the function of this uncharacterized protein, researchers should consider multiple complementary approaches:

• Subcellular localization studies: Using fluorescently tagged versions of the protein to determine its cellular distribution and potential interaction with specific organelles.

• Protein-protein interaction studies: Employing techniques such as co-immunoprecipitation, yeast two-hybrid, or proximity labeling approaches to identify binding partners.

• Gene knockout/knockdown experiments: CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown in appropriate cell lines to observe resulting phenotypes.

• Comparative genomics: Analysis across species to identify conserved domains that might suggest function.

• Structural analysis: X-ray crystallography or cryo-EM studies to determine three-dimensional structure that might provide functional insights .

What are the optimal storage conditions for the recombinant protein?

The recombinant Mouse C1orf95 homolog protein should be stored at -20°C for regular use and at -80°C for long-term storage. The protein is typically supplied as a lyophilized powder and should be reconstituted before use. For working aliquots, storage at 4°C is suitable for up to one week. Importantly, repeated freeze-thaw cycles should be avoided to prevent protein degradation and loss of activity .

What is the recommended protocol for reconstitution?

The recommended reconstitution protocol for the lyophilized protein includes:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom.

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

• Add glycerol to a final concentration of 5-50% (typically 50% is recommended) for stability.

• Aliquot into working volumes to avoid repeated freeze-thaw cycles.

• Store reconstituted protein at appropriate temperatures based on usage timeline (-20°C/-80°C for long-term; 4°C for short-term use up to one week) .

What buffer systems are compatible with this protein for functional studies?

The recombinant protein is supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0. For functional studies, compatible buffer systems would likely include:

• Tris-based buffers (pH 7.4-8.0)

• Phosphate buffers (pH 7.2-7.4)

• HEPES buffers (pH 7.0-7.6)

When conducting functional studies, researchers should consider incorporating:

• Physiological salt concentrations (e.g., 150 mM NaCl)

• Potential stabilizing agents (e.g., glycerol)

• Appropriate detergents for membrane protein solubility if removed from membranes

• Protease inhibitors to prevent degradation during extended experiments

How does the mouse C1orf95 homolog compare to human variants?

While detailed comparative analyses are not provided in the available search results, researchers investigating this protein should consider:

• Sequence homology analysis between mouse and human variants to identify conserved domains that might be functionally significant.

• Comparison of expression patterns in different tissues across species.

• Evolutionary conservation analysis to identify crucial structural or functional regions.

• Analysis of potential post-translational modifications that might differ between species.

This comparative approach may provide insights into functional significance and identify regions suitable for targeting in experimental manipulations .

What is currently understood about this protein's role in cellular signaling?

The current designation as an "uncharacterized membrane protein" indicates limited knowledge regarding its specific roles in cellular signaling. Based on its classification as a membrane protein, potential functions might include:

• Signal transduction across membranes

• Transport of molecules

• Cell-cell communication

• Structural roles in membrane organization

Researchers investigating this protein should consider its potential interactions with known signaling pathways by examining co-expression patterns, subcellular localization, and functional impacts of gene knockout/knockdown in relevant cellular models .

What experimental approaches can determine the topology of this membrane protein?

Determining the topology of membrane proteins like C1orf95 homolog requires specialized techniques:

• Protease protection assays: Exposing intact vesicles containing the protein to proteases to determine which regions are accessible.

• Fluorescence quenching experiments: Using environment-sensitive fluorophores attached to specific residues.

• Glycosylation mapping: Introducing artificial glycosylation sites to determine luminal versus cytoplasmic orientation.

• Cysteine scanning mutagenesis: Combining with thiol-reactive reagents to determine accessibility.

• Cryo-EM or X-ray crystallography: For high-resolution structural determination, though these approaches are challenging for membrane proteins.

The combination of multiple approaches provides the most reliable topology determination .

What quality control measures should be applied when working with this recombinant protein?

When working with Recombinant Mouse C1orf95 homolog protein, researchers should implement several quality control measures:

• Purity assessment: SDS-PAGE analysis to confirm protein purity (>90% as specified in product information).

• Identity confirmation: Western blotting using appropriate antibodies targeting either the protein itself or the His-tag.

• Structural integrity: Circular dichroism (CD) spectroscopy to assess secondary structure characteristics.

• Aggregation assessment: Size exclusion chromatography or dynamic light scattering to detect potential aggregation.

• Endotoxin testing: For experiments sensitive to bacterial endotoxin contamination, particularly for immunological studies .

What are common challenges when working with membrane proteins like C1orf95 homolog?

Membrane proteins present several distinct challenges in experimental settings:

• Solubility issues: Maintaining proper folding and solubility outside of their native membrane environment.

• Aggregation tendency: Prone to aggregation due to exposure of hydrophobic surfaces.

• Functional assessment: Difficulty in assessing functional activity without knowledge of native function.

• Structural stability: May require specific lipid environments or detergents to maintain native conformation.

• Expression challenges: Often express at lower levels than soluble proteins in recombinant systems.

To address these challenges, researchers should consider:

• Using mild detergents appropriate for membrane proteins

• Incorporating lipids or membrane mimetics in experimental buffers

• Employing gentle handling techniques to minimize protein denaturation

• Testing multiple buffer conditions to optimize stability .

What are potential research applications for this uncharacterized protein?

Despite its uncharacterized status, the Recombinant Mouse C1orf95 homolog protein may serve several research purposes:

• Antibody production: Generation of specific antibodies for localization and expression studies.

• Protein-protein interaction screening: Identification of binding partners to suggest functional pathways.

• Structural studies: Determination of three-dimensional structure to provide functional insights.

• Comparative studies: Understanding conserved functions across species.

• Drug target assessment: Evaluation as a potential therapeutic target once function is better understood .

How might researchers approach elucidating the biological function of this protein?

A systematic approach to determine the biological function might include:

• Bioinformatic analysis: Prediction of function based on sequence homology, conserved domains, and structural modeling.

• Expression profiling: Determination of tissue distribution and expression patterns under various conditions.

• Knockout/knockdown studies: Generation of cell lines or animal models lacking the protein to observe phenotypic changes.

• Overexpression studies: Examining effects of increased protein levels on cellular processes.

• Interaction network mapping: Identification of the protein's position within cellular interaction networks.

• Subcellular localization: Detailed analysis of the protein's distribution within cellular compartments.

This multi-faceted approach would provide complementary lines of evidence regarding the protein's biological role .

What methodological approaches are suitable for detecting this protein in experimental samples?

Detection of the Recombinant Mouse C1orf95 homolog protein can be accomplished through various methodologies:

• Western blotting: Using antibodies against the protein itself or the His-tag for detection in cell/tissue lysates.

• Immunofluorescence: Visualizing subcellular localization in fixed cells or tissues.

• ELISA: Quantitative detection in solution samples.

• Mass spectrometry: Identification and quantification in complex protein mixtures.

For the His-tagged recombinant version, additional detection options include:

• Ni-NTA conjugated detection reagents

• Anti-His antibodies

• Fluorescent Ni-NTA derivatives for direct visualization .

What experimental controls are essential when studying this uncharacterized protein?

When designing experiments involving the C1orf95 homolog protein, researchers should include these essential controls:

• Negative controls:

  • Samples lacking the protein of interest

  • Irrelevant proteins with similar characteristics (size, charge, tags)

  • Appropriate isotype controls for antibody-based experiments

• Positive controls:

  • Known amounts of purified recombinant protein

  • Well-characterized membrane proteins processed similarly

  • Samples with confirmed expression of the target protein

• Experimental validation controls:

  • Multiple detection methods to confirm findings

  • Dose-response relationships where applicable

  • Replication across different experimental conditions or models .