Recombinant Xenopus tropicalis Coiled-coil domain-containing protein 90B, mitochondrial (ccdc90b)

How conserved is ccdc90b across vertebrate species?

Analysis of sequence conservation indicates that ccdc90b is moderately conserved across vertebrate species. While specific conservation data for ccdc90b is limited in the available resources, comparative sequence analysis with similar proteins such as TACC family members in Xenopus shows approximately 50% identity across species, with functional domains showing higher conservation (up to 80% in conserved domains) .

What is the predicted cellular localization and function of ccdc90b?

Ccdc90b is primarily localized to mitochondria as indicated by its full name (Coiled-coil domain-containing protein 90B, mitochondrial). GO term prediction studies have successfully identified the cellular component location of CCDC90B using computational approaches such as I-TASSER/COFACTOR .

Functionally, while the specific role of ccdc90b in Xenopus tropicalis is not fully characterized in the provided resources, its mitochondrial localization suggests involvement in:

• Mitochondrial transport processes

• Mitochondrial organization or dynamics

• Potential roles in mitochondrial protein complexes

• Energy metabolism pathways

What are the optimal storage and handling conditions for recombinant ccdc90b?

Based on product information for recombinant ccdc90b, optimal storage conditions are:

• Storage buffer: Tris-based buffer with 50% glycerol, optimized for protein stability

• Long-term storage: -20°C or -80°C for extended preservation

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

• Avoid repeated freeze-thaw cycles as this may reduce protein activity and stability

For experiments requiring active protein, researchers should thaw aliquots on ice and maintain cold chain throughout experimental procedures.

What expression systems are suitable for producing recombinant ccdc90b?

While the search results don't specify the exact expression system used for the referenced recombinant ccdc90b, standard approaches for similar mitochondrial proteins include:

• Bacterial expression systems (E. coli):

  • Advantages: High yield, cost-effective

  • Limitations: May lack post-translational modifications

  • Recommended for structural studies or antibody production

• Eukaryotic expression systems:

  • Mammalian cell lines (e.g., HEK293T cells) for functional studies

  • Xenopus oocytes for expression of Xenopus proteins in a native-like environment

The choice should depend on experimental goals, required protein folding, and post-translational modifications needed for functional studies.

How can I perform loss-of-function analysis for ccdc90b in Xenopus tropicalis?

While specific protocols for ccdc90b are not detailed in the search results, general approaches for loss-of-function analysis in Xenopus include:

• Morpholino-based knockdown:

  • Design morpholinos targeting the ATG start site or splice sites

  • Inject into early embryos (1-2 cell stage)

  • Validate knockdown efficiency by Western blot

• CRISPR-Cas9 genome editing:

  • Design guide RNAs targeting early exons of ccdc90b

  • Validate editing efficiency by sequencing and protein expression analysis

  • Analyze phenotypes in F0 or establish knockout lines

• Dominant negative approaches:

  • Express truncated versions lacking specific functional domains

  • Analyze resulting phenotypes for insights into normal function

Each approach has advantages and limitations; selection should be based on specific research questions and available resources.

What methods are effective for identifying ccdc90b interaction partners?

To identify interaction partners of ccdc90b, affinity purification coupled with mass spectrometry (AP-MS) represents a powerful approach. Based on similar studies for other proteins:

• Experimental design:

  • Express tagged versions of ccdc90b (potential tags: Myc, Flag, or 2B8 as used for other proteins)

  • Perform immunoprecipitation using antibodies against the tags

  • Identify co-precipitated proteins by mass spectrometry

• Validation strategies:

  • Co-immunoprecipitation with candidate interactors

  • Proximity labeling approaches (BioID, APEX)

  • Co-localization studies using immunofluorescence

  • Functional validation through co-expression studies

For instance, a protocol similar to that used for C11orf52 could be adapted, where "transient transfection of plasmid DNA containing [protein] and three epitope tags (Myc, Flag, and 2B8) was performed using a Turbofect device," followed by cell lysis and immunoprecipitation .

How can I study ccdc90b localization within Xenopus cells?

To study subcellular localization of ccdc90b:

• Immunofluorescence approaches:

  • Use antibodies against native ccdc90b or epitope tags on recombinant protein

  • Co-stain with mitochondrial markers (e.g., MitoTracker)

  • Analyze using confocal microscopy

• Live-cell imaging with fluorescent fusion proteins:

  • Generate GFP- or mCherry-tagged ccdc90b constructs

  • Express in Xenopus embryonic cells

  • Monitor localization and dynamics in live cells

Similar approaches have been successful for studying other proteins in Xenopus cells, such as TACC1 where "GFP-TACC1 localized to the growing plus-ends of MTs in mesenchymal cells derived from the neural tube" .

What experimental approaches can determine if ccdc90b functions in mitochondrial transport?

To investigate potential roles in mitochondrial transport:

• Mitochondrial motility assays:

  • Knockdown or overexpress ccdc90b in Xenopus cells

  • Label mitochondria with fluorescent markers

  • Track mitochondrial movement using time-lapse microscopy

  • Analyze transport parameters (speed, distance, directionality)

• Mitochondrial fractionation:

  • Isolate mitochondria from ccdc90b-manipulated and control cells

  • Analyze mitochondrial protein composition by Western blot or proteomics

  • Identify changes in transport-related proteins

• Functional assays:

  • Measure mitochondrial membrane potential using fluorescent indicators

  • Assess mitochondrial calcium handling

  • Evaluate respiratory chain activity in ccdc90b-manipulated cells

Similar approaches have been used to study mitochondrial transport proteins in other contexts .

How can computational prediction tools enhance understanding of ccdc90b function?

Computational prediction represents a powerful approach to generate functional hypotheses about ccdc90b:

• GO term prediction using I-TASSER/COFACTOR:

  • This approach has successfully identified cellular component localization of CCDC90B

  • The method generates GO terms with associated confidence scores

  • Results can be filtered using specialized algorithms to improve precision

• Implementation approach:

  • Generate structural models of ccdc90b using I-TASSER

  • Use COFACTOR to predict GO terms based on structural similarity

  • Apply filtering algorithms to select specific GO terms with higher precision-recall scores

  • Validate predictions experimentally

Research has shown that "187 specific GO terms showed a higher av. precision-recall score at the least cellular component term compared to 2413 predicted GO terms" when using this approach for similar proteins .

What is known about the expression and regulation of ccdc90b during Xenopus development?

While specific information about ccdc90b expression patterns in Xenopus development is not detailed in the search results, approaches to study developmental expression include:

• RT-PCR analysis across developmental stages:

  • Similar to approaches used for TACC family proteins where "RT-PCR of Xenopus laevis cDNA libraries from various developmental time points and tissues" revealed expression patterns

  • Compare expression in neural, epidermal, and mesoendodermal tissues

• In situ hybridization:

  • Develop specific probes for ccdc90b mRNA

  • Analyze spatial expression patterns in embryos at different stages

• Protein expression analysis:

  • Western blotting of embryonic tissues at different stages

  • Immunohistochemistry to localize protein expression spatially

These approaches would provide insights into the developmental roles of ccdc90b, potentially revealing stage- or tissue-specific functions.

How does ccdc90b function compare between Xenopus and mammalian systems?

Comparative functional analysis between Xenopus and mammalian ccdc90b provides evolutionary insights:

• Sequence and structural comparison:

  • Align sequences to identify conserved domains and species-specific variations

  • Predict structural differences that might impact function

  • Identify conserved post-translational modification sites

• Cross-species functional rescue experiments:

  • Rescue ccdc90b knockdown in Xenopus with mammalian homologs

  • Quantify rescue efficiency to determine functional conservation

  • Identify domains required for conserved functions

• Interaction partner analysis:

  • Compare interaction networks between species

  • Identify conserved and species-specific interactors

  • Determine if molecular mechanisms are conserved across vertebrates

Similar comparative approaches have been applied to TACC proteins, where functional differences were observed between Xenopus and human systems despite sequence conservation .

What quality control measures should be employed when working with recombinant ccdc90b?

Rigorous quality control is essential for reliable research with recombinant proteins:

• Purity assessment:

  • SDS-PAGE with Coomassie staining to verify size and purity

  • Western blot with specific antibodies to confirm identity

  • Mass spectrometry for definitive identification

• Functional validation:

  • Activity assays appropriate to predicted function

  • Binding assays with known or predicted partners

  • Structural integrity assessment (circular dichroism, thermal shift assays)

• Batch-to-batch consistency:

  • Maintain detailed records of protein preparation and quality metrics

  • Consider aliquoting single preparations for longitudinal studies

  • Include positive controls in functional assays to normalize between batches