Recombinant Bovine Uncharacterized protein C17orf78 homolog

What are the recommended storage and handling protocols for the recombinant protein?

For optimal stability and activity maintenance, the following protocol is recommended:

• Store lyophilized powder at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use scenarios .

• For reconstitution, centrifuge the vial briefly before opening, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) and aliquot for storage at -20°C/-80°C .

• Working aliquots can be stored at 4°C for up to one week .

• Storage buffer typically consists of a Tris/PBS-based buffer with 6% trehalose or 50% glycerol at pH 8.0, optimized for protein stability .

• Avoid repeated freeze-thaw cycles as they can compromise protein integrity .

What expression systems are used for producing this recombinant protein?

Multiple expression systems can be utilized to produce Recombinant Bovine Uncharacterized protein C17orf78 homolog:

• E. coli: The most commonly used system, offering high yield and cost-effectiveness .

• Yeast: Alternative eukaryotic expression system that may provide different post-translational modifications .

• Baculovirus: Insect cell-based system useful for complex proteins requiring eukaryotic processing .

• Mammalian Cell: Provides the most native-like post-translational modifications but typically with lower yield .

• Cell-Free Expression: Allows for rapid production without cellular constraints .

Each system offers distinct advantages depending on the experimental requirements. The recombinant protein typically achieves ≥85-90% purity as determined by SDS-PAGE analysis .

How should I design experiments to investigate the subcellular localization of Bovine C17orf78 homolog?

Based on studies of related proteins such as human C17orf80, a systematic approach to localization studies might include:

• Immunofluorescence microscopy:

  • Express the recombinant protein fused to a fluorescent tag in bovine cells

  • Use antibodies against the His-tag or the protein itself

  • Co-stain with organelle markers (particularly mitochondrial markers, based on C17orf80 data)

  • Perform confocal microscopy analysis to determine precise subcellular distribution

• Cell fractionation:

  • Isolate subcellular compartments (cytosol, mitochondria, nucleus, etc.)

  • Analyze the presence of C17orf78 homolog in each fraction by Western blotting

  • Include appropriate fraction markers as controls

• Antibody accessibility assays:

  • Similar to techniques used for human C17orf80, employ selective membrane permeabilization

  • Use digitonin to permeabilize plasma membrane while keeping inner membranes intact

  • Add Triton X-100 to disrupt all cellular membranes

  • Compare staining patterns to determine membrane topology

• Bioinformatic prediction validation:

  • Test predicted transmembrane domains experimentally

  • Investigate potential membrane association mechanisms

Human C17orf80 studies revealed mitochondrial localization with specific association with mitochondrial nucleoids, suggesting a similar approach may be valuable for the bovine homolog .

What approaches can identify potential interaction partners of C17orf78 homolog?

To elucidate the functional network of this uncharacterized protein:

• Co-immunoprecipitation (Co-IP):

  • Leverage the His-tag for pulldown experiments from bovine cell extracts

  • Identify co-precipitating proteins by mass spectrometry

  • Validate key interactions with reverse Co-IP or Western blotting

• Proximity labeling:

  • Create fusion constructs with BioID or APEX2 enzymes

  • Express in bovine cells to biotinylate proximal proteins

  • Purify and identify biotinylated proteins by mass spectrometry

  • This approach was successful for human C17orf80, revealing nucleoid associations

• Yeast two-hybrid screening:

  • Use C17orf78 homolog as bait against a bovine cDNA library

  • Validate positive interactions with orthogonal methods

• Targeted interaction studies:

  • Based on C17orf80 findings, test interactions with mitochondrial nucleoid components

  • Investigate associations with mtDNA, TFAM, and DNA replication machinery

  • Assess if interactions persist after treatment with mtDNA replication inhibitors

• Cross-linking mass spectrometry:

  • Use chemical cross-linkers to stabilize transient protein-protein interactions

  • Map interaction interfaces at amino acid resolution

How can I assess the potential role of C17orf78 homolog in mitochondrial function?

Building on findings from human C17orf80 that demonstrated mitochondrial nucleoid association, a comprehensive approach might include:

• Genetic manipulation:

  • Generate knockdown or knockout bovine cell lines

  • Assess effects on:

    • Mitochondrial morphology (microscopy)

    • Membrane potential (fluorescent dyes)

    • Respiration (oxygen consumption measurements)

    • ATP production (luciferase assays)

    • mtDNA copy number and integrity (qPCR)

• Nucleoid association studies:

  • Colocalize C17orf78 homolog with nucleoid markers (TFAM, DNA)

  • Test if this colocalization persists after treatment with mtDNA replication inhibitors

  • As observed with human C17orf80, the protein may remain associated with clustered nucleoids after ethidium bromide or ddC treatment

• Mitochondrial gene expression analysis:

  • Measure mitochondrial transcript levels

  • Assess mitochondrial protein synthesis

  • Analyze respiratory complex assembly

• Stress response experiments:

  • Challenge cells with various stressors (oxidative stress, mtDNA depletion)

  • Monitor changes in C17orf78 homolog expression, localization, and interactions

What experimental approaches could determine if C17orf78 homolog has enzymatic activity?

To investigate potential enzymatic functions:

• Sequence and structural analysis:

  • Perform detailed motif searches and structural predictions

  • Identify potential catalytic residues or domains

  • Compare with known enzymes to generate testable hypotheses

• Generic enzyme activity screens:

  • Test recombinant protein with substrate libraries for common enzymatic activities

  • Screen for kinase, phosphatase, transferase, or hydrolase activities

  • Monitor substrate conversion using appropriate detection methods

• Nucleic acid-related activities:

  • Given the association of human C17orf80 with nucleoids, test for:

    • DNA/RNA binding (electrophoretic mobility shift assays)

    • Nuclease activity (using labeled substrates)

    • DNA repair functions (using damaged DNA templates)

    • Transcription or replication roles

• Structure-function studies:

  • Generate point mutations in predicted catalytic residues

  • Create truncation variants to isolate functional domains

  • Assess impact on activity and localization

How can I investigate potential disease associations of Bovine C17orf78 homolog?

Human C17orf80 has been linked to autism spectrum disorder and certain cancers . To explore potential disease associations of the bovine homolog:

• Expression analysis in pathological states:

  • Compare expression levels between healthy and diseased bovine tissues

  • Focus on tissues with known pathologies that might involve mitochondrial dysfunction

  • Use quantitative PCR, Western blotting, or immunohistochemistry

• Genetic association studies:

  • Identify single nucleotide polymorphisms (SNPs) in bovine populations

  • Correlate genetic variations with disease phenotypes or production traits

  • Perform targeted sequencing in animals with relevant disorders

• Functional studies in disease models:

  • Create cellular models mimicking disease conditions

  • Assess changes in C17orf78 homolog expression, localization, or interactions

  • Determine if modulating the protein levels affects disease phenotypes

• Comparative studies with human disorders:

  • Investigate functional similarities between bovine C17orf78 homolog and human C17orf80

  • Assess if findings in bovine models could inform human mitochondrial diseases

What approaches can resolve data inconsistencies in C17orf78 homolog research?

When facing contradictory results:

• Protein quality assessment:

  • Verify protein integrity using multiple techniques (SDS-PAGE, mass spectrometry)

  • Assess proper folding using biophysical methods

  • Test different protein preparations (varying tags, expression systems)

• Experimental condition optimization:

  • Systematically vary buffer conditions, pH, salt concentration

  • Test multiple cell types or tissue sources

  • Include appropriate positive and negative controls

• Technical approach diversification:

  • Validate findings using orthogonal techniques

  • For localization studies, combine microscopy with biochemical fractionation

  • For interaction studies, use multiple complementary methods

• Statistical rigor enhancement:

  • Increase biological and technical replicates

  • Perform power analysis to determine adequate sample sizes

  • Apply appropriate statistical tests and corrections

How might new technologies advance our understanding of C17orf78 homolog function?

Cutting-edge approaches that could provide new insights include:

• AlphaFold and structural biology:

  • Generate high-confidence 3D structural models

  • Use predicted structures to identify potential functional sites

  • Design targeted mutations based on structural insights

• CRISPR-based approaches:

  • Perform genome-wide CRISPR screens to identify genetic interactions

  • Use CRISPR base editing for precise mutagenesis

  • Create cellular models with endogenously tagged protein for physiological studies

• Advanced imaging:

  • Apply super-resolution microscopy for detailed localization

  • Use live-cell imaging to track protein dynamics

  • Implement correlative light and electron microscopy

• Multi-omics integration:

  • Combine proteomics, transcriptomics, and metabolomics data

  • Use systems biology approaches to place C17orf78 homolog in biological networks

  • Develop predictive models of protein function

What experimental design would best determine if bovine C17orf78 homolog shares functional similarity with human C17orf80?

To establish functional homology:

• Complementation studies:

  • Knock down or knock out human C17orf80 in human cells

  • Express bovine C17orf78 homolog and assess rescue of phenotypes

  • Analyze if the bovine protein localizes to mitochondrial nucleoids like its human counterpart

• Comparative interaction studies:

  • Identify interaction partners of both proteins using identical methods

  • Compare interactomes to identify shared and unique interactions

  • Focus on nucleoid components that interact with human C17orf80

• Parallel phenotypic analysis:

  • Generate knockout cell lines for both proteins

  • Perform side-by-side phenotypic characterization

  • Use identical assays to measure effects on mitochondrial function and mtDNA maintenance

• Domain swap experiments:

  • Create chimeric proteins containing domains from both human and bovine proteins

  • Test functionality in localization and interaction assays

  • Identify domains responsible for specific functions