Recombinant Danio rerio UPF0767 protein C1orf212 homolog (si:ch73-31g16.1, zgc:103571)

What is the molecular identity of Recombinant Danio rerio UPF0767 protein C1orf212 homolog?

Recombinant Danio rerio UPF0767 protein C1orf212 homolog (si:ch73-31g16.1, zgc:103571) is a transmembrane protein derived from zebrafish with UniProt accession number Q5BKW8 . The protein is also known by its target name "smim12" and represents the full-length version of the native protein as expressed recombinantly in an E. coli expression system . This recombinant version features an N-terminal 10xHis tag that facilitates purification and detection in experimental settings, while maintaining the complete amino acid sequence of the native protein . The protein consists of 91 amino acids, covering the expression region 1-91, which represents the complete coding sequence of this relatively small transmembrane protein .

What is the amino acid sequence of the protein and what structural features does it contain?

The amino acid sequence of Recombinant Danio rerio UPF0767 protein C1orf212 homolog is: MWPIFWTAMRTYAPYVTFPVAFVVGAVGYHLEWFIRGTPNTPGEERGIAELREDRKLEEL NGRDSTQVLSLKDKLEFTPRAVLERNRAVKS . This 91-amino acid sequence contains the characteristic features of a transmembrane protein, with hydrophobic regions that likely span the cellular membrane. The sequence contains a mixture of hydrophobic residues (such as tryptophan, phenylalanine, valine, and leucine) that typically constitute the membrane-spanning domains, as well as charged and polar residues that likely reside in the cytoplasmic and extracellular domains. Structural prediction algorithms suggest the presence of at least one transmembrane domain, consistent with its classification as a transmembrane protein in the product specifications . The presence of the N-terminal 10xHis tag in the recombinant version provides an additional functional element that does not exist in the native protein but serves as a crucial tool for laboratory applications.

How is the recombinant protein produced and what expression system is used?

The recombinant Danio rerio UPF0767 protein C1orf212 homolog is produced using an in vitro E. coli expression system, which is one of the most common and efficient platforms for recombinant protein production in research settings . The production process likely involves cloning the coding sequence of the protein into an expression vector containing the necessary regulatory elements for protein expression in E. coli, including a promoter sequence, ribosome binding site, and sequences encoding the N-terminal 10xHis tag. Following transformation into E. coli, the bacteria are cultured under controlled conditions to induce protein expression, after which the cells are harvested and lysed to release the recombinant protein. The protein is then purified using affinity chromatography, taking advantage of the N-terminal 10xHis tag which binds strongly to nickel or cobalt resin columns. After purification, the protein may undergo additional processing steps such as concentration, buffer exchange, and quality control testing before being formulated in its final form as either a liquid or lyophilized powder.

What are the optimal storage conditions for maintaining protein stability?

For optimal stability of Recombinant Danio rerio UPF0767 protein C1orf212 homolog, the recommended storage temperature is -20°C, with extended storage preferably at -20°C or -80°C to prevent degradation over time . The protein should be stored in appropriate aliquots to minimize freeze-thaw cycles, as repeated freezing and thawing can significantly compromise protein integrity and activity . Working aliquots can be stored at 4°C for up to one week, but longer periods at this temperature are not recommended due to potential degradation . The shelf life of the protein varies depending on its formulation: the liquid form generally maintains stability for approximately 6 months when stored at -20°C/-80°C, while the lyophilized form exhibits greater stability with a shelf life of approximately 12 months at the same temperature range . Factors affecting shelf life include the buffer composition, storage temperature, and the intrinsic stability of the protein itself, which can vary between different preparations and batches .

How should researchers reconstitute lyophilized Recombinant Danio rerio UPF0767 protein for experimental use?

To properly reconstitute lyophilized Recombinant Danio rerio UPF0767 protein C1orf212 homolog, researchers should first briefly centrifuge the vial to ensure all content is at the bottom, minimizing product loss during the opening process . The lyophilized protein should be reconstituted using deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL, ensuring complete dissolution by gentle mixing rather than vigorous vortexing which could denature the protein . For long-term storage of reconstituted protein, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) to prevent freeze damage during storage at -20°C/-80°C . The reconstitution buffer should be compatible with the downstream applications and should maintain the protein in its native conformation; while the product comes in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, researchers should consider buffer exchange if their experimental conditions require different buffer systems . After reconstitution, the solution should be gently mixed and allowed to stand for a few minutes at room temperature to ensure complete solubilization before aliquoting to minimize freeze-thaw cycles.

What quality control methods should be employed to verify protein integrity before experimental use?

Quality control of Recombinant Danio rerio UPF0767 protein C1orf212 homolog should begin with visual inspection for any obvious signs of precipitation or discoloration that might indicate degradation or contamination. SDS-PAGE analysis should be performed to verify protein purity, with the expectation that the product should demonstrate greater than 90% purity as typically determined for commercial recombinant proteins . Western blotting using antibodies against either the His-tag or the protein itself can confirm the identity of the protein and detect any degradation products that may have formed during storage. Functional assays relevant to the protein's known or predicted biological activities should be conducted to ensure that the protein has maintained its proper folding and activity; these might include binding assays, enzymatic activity tests, or cell-based functional assays depending on the known properties of the protein. For transmembrane proteins like UPF0767 protein C1orf212 homolog, additional tests such as circular dichroism spectroscopy might be valuable to assess secondary structure integrity, particularly if the protein's function depends on specific structural conformations within the membrane environment.

How can researchers effectively incorporate this protein into zebrafish developmental studies?

When incorporating Recombinant Danio rerio UPF0767 protein C1orf212 homolog into zebrafish developmental studies, researchers should begin by establishing appropriate delivery methods that ensure the protein reaches its intended cellular targets. Microinjection techniques can be used to deliver the recombinant protein directly into zebrafish embryos at early developmental stages, allowing for the study of its effects on embryonic development and organogenesis. Time-course experiments should be designed to track the effects of the protein at different developmental stages, with careful documentation of morphological changes, gene expression patterns, and phenotypic outcomes. Immunohistochemistry using antibodies against the His-tag or the protein itself can help localize the exogenously introduced protein within tissues and cells, providing insights into its distribution and potential sites of action. Control experiments are essential and should include both negative controls (buffer-only injections) and specificity controls (injections of irrelevant proteins with similar characteristics) to distinguish specific effects from non-specific consequences of protein introduction.

What methodological approaches are most effective for studying transmembrane protein function in cell culture systems?

For studying the function of Recombinant Danio rerio UPF0767 protein C1orf212 homolog in cell culture systems, researchers should select appropriate cell lines that either naturally express complementary components of the signaling pathways involved or can be engineered to do so. Transfection or transduction methods should be optimized to achieve efficient expression of the recombinant protein in the cell membrane, potentially using lipid-based transfection reagents or viral vectors depending on the cell type. Cell membrane fractionation techniques can be employed to verify proper localization of the protein to the membrane compartment, followed by functional assays to assess the protein's effects on cellular processes such as signal transduction, ion flux, or membrane permeability. Co-immunoprecipitation experiments using the His-tag as a handle can help identify interaction partners, providing insights into the protein's role in cellular signaling networks. Live-cell imaging using fluorescently tagged versions of the protein can offer dynamic information about its localization, trafficking, and potential conformational changes in response to stimuli.

How should investigators design experiments to elucidate the functional role of UPF0767 protein C1orf212 homolog in zebrafish?

A comprehensive experimental strategy to elucidate the functional role of UPF0767 protein C1orf212 homolog should begin with gene expression analysis using techniques such as qRT-PCR, in situ hybridization, or RNA-Seq to characterize the temporal and spatial expression patterns of the endogenous gene during zebrafish development. Loss-of-function studies using morpholinos, CRISPR-Cas9 genome editing, or dominant-negative approaches can reveal the consequences of reduced or abolished protein function, while gain-of-function experiments using mRNA injection or transgenic overexpression can demonstrate the effects of increased protein levels. Rescue experiments, in which the recombinant protein is introduced into loss-of-function models, can validate the specificity of observed phenotypes and potentially reveal structure-function relationships if modified versions of the protein are used. Careful phenotypic analysis should include not only gross morphological assessments but also molecular markers of relevant developmental processes, tissue-specific functional assays, and behavioral studies if appropriate. Integration of findings with existing knowledge about similar proteins in other species can provide evolutionary context and potentially reveal conserved functional aspects of this protein family.

How does the N-terminal His-tag affect the functionality and experimental utility of the protein?

The N-terminal 10xHis-tag on Recombinant Danio rerio UPF0767 protein C1orf212 homolog serves as a crucial tool for purification and detection but may potentially influence the protein's functionality in experimental settings . The tag's position at the N-terminus could affect protein folding, particularly if the N-terminal region plays a role in the protein's native conformation or interaction with other molecules. Researchers should consider conducting parallel experiments with tagged and untagged versions of the protein when possible, or implementing tag removal using specific proteases if the experimental design requires the native protein structure. For immunodetection purposes, the His-tag provides a universal epitope that can be recognized by anti-His antibodies, circumventing the need for protein-specific antibodies which may not be readily available for this relatively uncharacterized protein. In protein-protein interaction studies, controls should be included to ensure that observed interactions are not mediated by the His-tag itself rather than the protein of interest, possibly by using alternative tags or tag-free approaches as verification methods.

What are the potential mechanisms of action for UPF0767 protein C1orf212 homolog in cellular processes?

As a transmembrane protein, Recombinant Danio rerio UPF0767 protein C1orf212 homolog may participate in various cellular processes including signal transduction, membrane transport, cell adhesion, or structural organization of cellular compartments. The protein's classification in the UPF (Uncharacterized Protein Family) category indicates that its precise function remains to be fully elucidated, presenting both challenges and opportunities for novel discoveries . Sequence analysis and structural predictions suggest potential functional domains that might be involved in protein-protein interactions, ligand binding, or enzymatic activities, which can guide hypothesis generation for experimental testing. Comparative genomics approaches, examining orthologous proteins in other species, may provide insights into conserved functions that have been better characterized in other model organisms. Systematic screening for interaction partners using techniques such as yeast two-hybrid, protein microarrays, or proximity labeling followed by mass spectrometry can identify potential binding partners that might suggest functional pathways involving this protein. Integration of experimental data with bioinformatics predictions can help construct working models of the protein's potential roles in zebrafish cellular physiology.

How can researchers utilize this protein in comparative studies across species?

Comparative studies utilizing Recombinant Danio rerio UPF0767 protein C1orf212 homolog can provide valuable insights into evolutionary conservation and divergence of protein function across species. Sequence alignment with homologous proteins from other vertebrates, including the human C1orf212 protein, can identify conserved domains that likely represent functionally important regions maintained through evolutionary pressure. Functional complementation experiments, in which the zebrafish protein is expressed in cells or organisms where the endogenous homolog has been knocked out, can determine whether the zebrafish protein can rescue the loss-of-function phenotype, indicating functional conservation. Cross-species binding assays can determine whether the zebrafish protein can interact with partners of its homologs in other species, providing information about the conservation of interaction interfaces and signaling pathways. Structural studies comparing the three-dimensional conformations of homologous proteins across species can reveal conserved structural features that may be essential for function, as well as species-specific structural adaptations that might reflect specialized functions. Integration of findings with human disease-associated variations in homologous proteins can potentially identify zebrafish models relevant to human pathologies, bridging basic research with translational applications.

What are common challenges in working with transmembrane proteins like UPF0767 protein C1orf212 homolog?

Working with transmembrane proteins like Recombinant Danio rerio UPF0767 protein C1orf212 homolog presents several challenges that researchers should anticipate and address in their experimental design. Solubility issues are common, as transmembrane proteins contain hydrophobic domains that can lead to aggregation or precipitation in aqueous solutions; using appropriate detergents or lipid environments can help maintain the protein in a soluble and functional state. Maintaining the native conformation is particularly challenging for transmembrane proteins, as their structure is stabilized by the lipid bilayer in vivo; reconstitution into liposomes, nanodiscs, or other membrane mimetics may be necessary for certain functional studies. Protein degradation can occur rapidly for transmembrane proteins outside their native environment, necessitating careful handling, the use of protease inhibitors, and appropriate storage conditions to preserve integrity. Non-specific binding to laboratory plasticware due to the hydrophobic nature of these proteins can result in significant loss of material during experimental procedures; pre-coating containers with non-ionic detergents or proteins like BSA can minimize this issue. Functional assays for transmembrane proteins often require specialized setups that can recreate membrane environments, such as lipid bilayer electrophysiology systems, surface plasmon resonance platforms with lipid capture, or cell-based assays that allow for proper membrane integration.

How can researchers troubleshoot experiments when unexpected results occur?

When encountering unexpected results in experiments with Recombinant Danio rerio UPF0767 protein C1orf212 homolog, researchers should implement a systematic troubleshooting approach. First, verify the integrity and purity of the protein using SDS-PAGE, western blotting, or mass spectrometry to ensure that degradation or contamination is not the source of unexpected results. Conduct careful controls at each step of the experimental process, including positive and negative controls, vehicle controls, and specificity controls using unrelated proteins with similar characteristics. Examine experimental conditions such as buffer composition, pH, temperature, and ionic strength, as transmembrane proteins are particularly sensitive to these parameters and may require optimization beyond standard protocols. Consider potential interaction with experimental materials such as plasticware, filters, or other components that might adsorb or inactivate the protein. If the protein is being expressed in a heterologous system, verify proper expression, localization, and post-translational modifications using appropriate cellular or biochemical assays, as improper processing could lead to altered functionality.

What quality control data should be reported in research publications involving this protein?

When publishing research involving Recombinant Danio rerio UPF0767 protein C1orf212 homolog, comprehensive quality control data should be reported to ensure reproducibility and reliability of the findings. Detailed information about the protein source should be provided, including the expression system, purification method, tag information, and any commercial supplier information with catalog numbers . Purity assessment data should be included, typically in the form of SDS-PAGE images or chromatography profiles, with quantification of purity percentage and any detected impurities or degradation products. Identity confirmation should be documented through methods such as western blotting, mass spectrometry, or N-terminal sequencing to verify that the protein is indeed the expected UPF0767 protein C1orf212 homolog. Functional validation data relevant to the protein's known or expected activities should be presented to demonstrate that the protein preparation used in the study maintained its biological activity. Storage and handling conditions should be clearly described, including buffer composition, protein concentration, storage temperature, and any special handling procedures that were employed to maintain protein stability and functionality . Inclusion of this quality control information not only supports the validity of the reported findings but also enables other researchers to properly replicate and build upon the published work.

What are promising research areas for further characterization of UPF0767 protein C1orf212 homolog?

Future research on Recombinant Danio rerio UPF0767 protein C1orf212 homolog could productively focus on several promising areas. Structural biology approaches, including X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy, could elucidate the three-dimensional structure of the protein, providing insights into its functional domains and potential binding interfaces. Systematic interactome mapping using proteomics approaches could identify binding partners and position the protein within cellular signaling networks, potentially revealing its functional role through its associations. Development of conditional knockout or knockin zebrafish models using CRISPR-Cas9 technology would allow for temporal and tissue-specific manipulation of the protein's expression, enabling detailed analysis of its role in development and physiology. High-throughput screening for small molecule modulators of the protein's activity could generate valuable tool compounds for functional studies and potentially identify therapeutic leads if the protein or its human homolog is implicated in disease processes. Multi-omics integration, combining transcriptomics, proteomics, and metabolomics data from models with altered expression of the protein, could provide a systems-level understanding of its biological impact and identify downstream effector pathways.

How might research on this protein contribute to understanding human disease mechanisms?

Research on Recombinant Danio rerio UPF0767 protein C1orf212 homolog has potential implications for understanding human disease mechanisms through several avenues. Comparative genomic analysis could identify human orthologs and paralogs with shared functions, potentially linking the zebrafish protein to human proteins with known disease associations . Zebrafish models with altered expression of the protein could serve as valuable tools for studying the developmental and physiological processes that, when disrupted in humans, contribute to disease pathogenesis. If the human ortholog is implicated in specific disorders, the zebrafish protein could be used in functional studies to characterize disease-associated variants, potentially in complementation experiments where human variants are tested for their ability to rescue zebrafish phenotypes. Drug discovery efforts targeting this protein family could utilize the zebrafish system as an in vivo model for early-stage screening and validation of candidate therapeutic compounds. Integration of findings with human genetic data, such as genome-wide association studies or exome sequencing results, could potentially identify previously unrecognized connections between this protein family and human disease susceptibility or progression.

What emerging technologies might enhance research capabilities for studying transmembrane proteins like UPF0767 protein C1orf212 homolog?

Emerging technologies offer exciting opportunities to advance research on transmembrane proteins like Recombinant Danio rerio UPF0767 protein C1orf212 homolog. Single-molecule techniques, including single-molecule FRET or atomic force microscopy, can provide unprecedented insights into protein dynamics, conformational changes, and interaction kinetics at the individual molecule level. Advanced microscopy methods such as super-resolution microscopy or lattice light-sheet microscopy enable visualization of protein localization and trafficking with nanometer precision in living cells, potentially revealing dynamic aspects of the protein's function. Microfluidic organ-on-chip platforms incorporating zebrafish cells or tissues could create physiologically relevant environments for studying the protein's function in tissue contexts that better recapitulate in vivo conditions. CRISPR-based technologies beyond gene editing, such as CRISPRi, CRISPRa, or base editing, offer precise manipulation of gene expression or protein sequence without complete gene knockout, enabling nuanced studies of protein function. Integration of machine learning approaches with experimental data could accelerate hypothesis generation and guide experimental design by predicting protein-protein interactions, functional domains, or phenotypic outcomes based on sequence or structural features, potentially revealing non-obvious aspects of the protein's biology that merit targeted investigation.