Recombinant Dog UPF0767 protein C1orf212 homolog

How is SMIM12/C1orf212 evolutionarily conserved across species?

While specific evolutionary data for C1orf212 is limited in current literature, the related C1ORF112 protein demonstrates significant evolutionary conservation across vertebrates, with homologs present in some invertebrates, plants, and single-celled microorganisms . Comparative analysis of C1orf212 would likely follow similar methodological approaches, utilizing sensitive HMM-HMM comparison methods like those used for C1ORF112. Researchers investigating evolutionary conservation should employ multiple sequence alignment tools (MUSCLE, CLUSTAL Omega) followed by phylogenetic analyses using maximum likelihood or Bayesian methods. Conservation mapping using tools like ConSurf can identify functionally important residues maintained through evolutionary pressure, particularly focusing on potential binding interfaces that might be more conserved than exterior surfaces.

What expression systems are optimal for producing Recombinant Dog UPF0767 protein C1orf212 homolog?

E. coli expression systems have been successfully used to produce Recombinant Dog UPF0767 protein C1orf212 homolog . For optimal expression, researchers should consider the following methodological approach: (1) Codon optimization of the gene sequence for the expression host; (2) Selection of an appropriate vector with an inducible promoter (e.g., T7 or tac); (3) Testing multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express) to identify optimal expression conditions; (4) Optimization of induction parameters including temperature (typically lowered to 16-25°C for proper folding), IPTG concentration (0.1-1.0 mM), and induction duration (4-24 hours). For challenging expressions, alternative systems such as mammalian (HEK293, CHO) or insect cell (Sf9, Hi5) platforms should be considered, particularly if post-translational modifications are critical for functional studies.

What is the most effective purification strategy for His-tagged Recombinant Dog UPF0767 protein C1orf212 homolog?

The most effective purification strategy for His-tagged Recombinant Dog UPF0767 protein C1orf212 homolog involves a multi-step approach. Begin with immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, employing a stepwise imidazole gradient (10-250 mM) for elution . For proteins expressed in E. coli as inclusion bodies, start with inclusion body isolation followed by solubilization in 6-8 M urea or guanidine hydrochloride, then perform refolding via dialysis against decreasing concentrations of denaturant . After initial IMAC purification, employ size exclusion chromatography (Superdex 75/200) as a polishing step to remove aggregates and ensure monodispersity. Protein purity should be assessed by SDS-PAGE (aim for >90% purity), and identity confirmed by western blotting and/or mass spectrometry. For storage, the purified protein can be lyophilized or stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0, with 50% glycerol for long-term storage at -20°C/-80°C .

How can researchers validate the functional integrity of purified Recombinant Dog UPF0767 protein C1orf212 homolog?

Validating functional integrity requires multiple analytical approaches. First, assess structural integrity through circular dichroism and thermal shift assays to confirm proper folding. Second, conduct binding assays to identify interaction partners, as proteins in the C1ORF family often function through protein-protein interactions . For this, consider using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or pull-down assays with potential binding partners identified through bioinformatic prediction. Third, since C1ORF family proteins may participate in DNA replication and repair pathways, functional assays could include DNA binding assays (EMSA) and assessment of effects on cell cycle progression in transfected cells. Finally, develop specific antibody-based detection methods to monitor the protein in cellular contexts, confirming subcellular localization through immunofluorescence microscopy, which can provide insights into potential function.

What analytical methods should be used to confirm protein identity and purity?

A comprehensive analytical pipeline should include multiple orthogonal techniques. Begin with SDS-PAGE to assess size and purity, aiming for >90% purity . Western blotting using anti-His antibodies confirms identity of the tagged protein. For definitive identification, employ mass spectrometry analysis, specifically peptide mass fingerprinting after tryptic digestion and LC-MS/MS for sequence verification. N-terminal sequencing can confirm the absence of unexpected processing. Analytical size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) provides information on oligomeric state and monodispersity. Dynamic light scattering (DLS) should be used to assess aggregation potential. Functional homogeneity can be evaluated through activity assays if known functions are established. For batch-to-batch consistency, establish a reference standard and compare new preparations using a subset of these analytical methods to ensure reproducibility in downstream experiments.

What are the cellular functions of SMIM12/C1orf212 homolog and how can they be investigated?

While specific functions of SMIM12/C1orf212 remain incompletely characterized, insights can be drawn from studies of related proteins. By analogy to C1ORF112, it may participate in DNA replication, damage repair pathways, or cell cycle regulation . To investigate cellular functions, researchers should employ a multi-faceted approach: (1) Gene silencing using siRNA targeting SMIM12 followed by phenotypic analysis, including cell cycle distribution, proliferation, and response to DNA damage; (2) Identification of interaction partners through co-immunoprecipitation followed by mass spectrometry or yeast two-hybrid screening; (3) Localization studies using fluorescently-tagged protein to determine subcellular distribution under normal and stress conditions; (4) Transcriptome analysis after gene knockdown or overexpression to identify affected pathways; (5) Chromatin immunoprecipitation studies if DNA binding is suspected. Functional redundancy should be considered when interpreting knockdown phenotypes, and validation across multiple cell lines is essential for establishing conserved functions.

How can researchers design experiments to investigate potential roles of C1orf212 homolog in DNA repair pathways?

Given the potential association of related proteins with DNA repair mechanisms , design a comprehensive experimental strategy: (1) Subject cells expressing or depleted of C1orf212 to various DNA damaging agents (UV, ionizing radiation, hydroxyurea, cisplatin) and assess survival, cell cycle checkpoints, and DNA damage markers (γH2AX foci); (2) Analyze recruitment of the protein to sites of DNA damage using laser microirradiation coupled with live-cell imaging of fluorescently-tagged protein; (3) Assess interaction with known DNA repair factors through co-immunoprecipitation or proximity ligation assays; (4) Measure repair pathway efficiency using reporter assays for homologous recombination, non-homologous end joining, and mismatch repair; (5) Generate domain mutants to identify regions critical for DNA damage response functions. Consider experimental timing carefully, as some repair functions may be early (sensing) or late (resolution) in the damage response. Controls should include established DNA repair proteins with known functions in the pathways under investigation.

What cell types and model systems are most appropriate for studying SMIM12/C1orf212 homolog?

Selection of appropriate model systems should be guided by expression patterns and research questions. For in vitro studies, begin with cell lines expressing detectable levels of endogenous SMIM12/C1orf212 (identified through mining expression databases like GTEx or Human Protein Atlas). Cancer cell lines may be particularly relevant given the association of related C1ORF proteins with cancer pathways . Consider creating stable cell lines with inducible expression or knockout of the gene using CRISPR-Cas9. For in vivo studies, knockout mouse models would be valuable, though careful phenotypic analysis is required as functional redundancy may mask effects. Importantly, when studying the dog homolog, canine-derived cell lines should be included for species-specific functions. Developmental model systems (zebrafish, Xenopus) may be valuable if embryonic functions are suspected. For each model system, validate antibodies or expression constructs thoroughly, and consider cross-species functionality when interpreting results.

How might post-translational modifications affect the function of Recombinant Dog UPF0767 protein C1orf212 homolog?

Post-translational modifications (PTMs) can significantly impact protein function, localization, and interactions. For Recombinant Dog UPF0767 protein C1orf212 homolog, researchers should consider: (1) Identification of potential PTM sites through bioinformatic prediction tools (NetPhos, SUMOplot, UbPred); (2) Comparison of bacterially-expressed recombinant protein with protein isolated from mammalian cells using mass spectrometry to identify native modifications; (3) Generation of PTM-mimetic mutants (e.g., phosphomimetic S/T to D/E substitutions) to assess functional impacts; (4) Treatment of cells with PTM inhibitors or stimulators to determine effects on protein function and localization; (5) Development of PTM-specific antibodies to track modification status under different cellular conditions. Particularly relevant may be phosphorylation events in response to cell cycle progression or DNA damage, if the protein functions in these contexts like related family members . Consider that recombinant protein expressed in E. coli will lack eukaryotic PTMs, potentially affecting functional studies.

What are the challenges in studying protein-protein interactions involving C1orf212 homolog and how can they be overcome?

Studying protein-protein interactions (PPIs) for this protein presents several challenges. First, transient or weak interactions may be difficult to capture with traditional approaches. To overcome this, implement stabilization strategies such as chemical crosslinking prior to immunoprecipitation or use proximity-dependent labeling methods (BioID, APEX). Second, the small size of the protein (92aa) may limit epitope availability for antibody-based techniques. Consider using larger fusion tags (HaloTag, SNAP-tag) that can be targeted with high-affinity ligands. Third, bacterially-expressed recombinant protein may lack critical PTMs necessary for interactions. Complement bacterial expression with mammalian cell expression systems. Fourth, unknown physiological conditions for optimal binding may hinder successful PPI detection. Screen multiple buffer conditions (varying salt, pH, and detergents) in binding assays. Finally, implement complementary techniques including co-immunoprecipitation, yeast two-hybrid, mammalian two-hybrid, fluorescence resonance energy transfer (FRET), and split-luciferase complementation assays to build confidence in identified interactions through orthogonal validation.

What bioinformatic approaches can predict structure-function relationships for understudied proteins like C1orf212 homolog?

For understudied proteins like C1orf212 homolog, bioinformatic approaches offer valuable insights into structure-function relationships. First, employ sensitive sequence comparison methods such as HMM-HMM searches using tools like HHpred, which successfully identified structural similarities between C1ORF112 and alpha helix-rich structures like importin beta . Second, use protein structure prediction tools (AlphaFold2, RoseTTAFold) to generate structural models, followed by molecular dynamics simulations to assess stability and conformational flexibility. Third, map sequence conservation onto structural models using tools like ConSurf to identify functional surfaces, as demonstrated for C1ORF112 where the inner curved surface showed higher conservation . Fourth, predict functional sites through integration of structural and evolutionary data using tools like COACH, COFACTOR, or ProFunc. Fifth, construct protein-protein interaction networks through database mining and co-expression analysis, which has proven valuable for related proteins . Finally, perform molecular docking simulations with predicted interaction partners or substrates. These computational predictions should guide experimental design, with each prediction systematically tested through mutation of key residues identified through bioinformatic analysis.

What are common pitfalls in recombinant expression of C1orf212 homolog and how can they be addressed?

Recombinant expression of C1orf212 homolog may encounter several challenges. First, insolubility and inclusion body formation in E. coli expression systems can be addressed by lowering induction temperature (16-20°C), reducing inducer concentration, using solubility-enhancing fusion partners (SUMO, MBP, TrxA), or switching to specialized E. coli strains (Arctic Express, Rosetta-gami). Second, protein instability can be mitigated by optimizing buffer conditions through thermal shift assays, including stabilizing additives like trehalose , and screening detergents if membrane association is suspected. Third, low expression levels may require codon optimization, stronger promoters, or alternative expression systems (mammalian, insect cells). Fourth, proteolytic degradation can be managed by adding protease inhibitors throughout purification and testing truncated constructs to identify stable domains. Fifth, loss of activity after purification might necessitate careful refolding protocols, particularly when recovering protein from inclusion bodies . Establish quality control metrics at each purification stage, incorporating activity assays where possible, and consider functional surfaces identified through bioinformatic analysis when designing expression constructs.

How should researchers design knockdown experiments to study SMIM12/C1orf212 function?

Designing effective knockdown experiments requires careful consideration of several factors. First, select appropriate knockdown tools: siRNA targeting SMIM12/C1orf212 for transient depletion, shRNA for stable depletion, or CRISPR-Cas9 for complete knockout. Second, validate knockdown efficiency using multiple approaches: RT-qPCR to measure mRNA levels, western blotting to confirm protein reduction, and include rescue experiments with RNAi-resistant constructs to establish specificity. Third, implement proper controls: non-targeting siRNA/shRNA, mock transfections, and knockdown of related genes to assess specificity of observed phenotypes. Fourth, optimize timing of analysis, as acute versus chronic depletion may yield different phenotypes due to compensatory mechanisms. Fifth, perform phenotypic analyses across multiple cellular processes, prioritizing pathways implicated through bioinformatic analysis or studies of related proteins, such as DNA replication and repair . Sixth, consider combinatorial knockdowns if functional redundancy is suspected. Finally, perform knockdowns in multiple cell types to identify cell-specific dependencies, as the requirement for SMIM12/C1orf212 function may vary across tissues.

What considerations are important when developing antibodies against Recombinant Dog UPF0767 protein C1orf212 homolog?

Developing effective antibodies against this protein requires strategic planning. First, select optimal antigens: use full-length recombinant protein for polyclonal antibodies but choose unique, accessible epitopes (avoiding transmembrane regions if present) for monoclonal antibody development. Second, consider cross-reactivity requirements: determine whether species-specific or cross-reactive antibodies are needed based on experimental goals, and perform sequence alignments to identify conserved versus divergent regions. Third, validate antibody specificity through multiple approaches: western blotting against recombinant protein and endogenous protein, testing in knockout/knockdown systems, peptide competition assays, and immunoprecipitation followed by mass spectrometry. Fourth, characterize application suitability: test antibodies in all intended applications (western blot, immunoprecipitation, immunofluorescence, flow cytometry, ChIP) as performance may vary. Fifth, establish reproducibility: compare multiple antibody lots and include appropriate positive and negative controls in all experiments. For challenging targets, consider alternative affinity reagents such as nanobodies or aptamers, which may offer advantages for recognizing specific conformational states or accessing sterically hindered epitopes.