Recombinant Drosophila melanogaster MOXD1 homolog 2 (olf413)
Description
Introduction to Recombinant Drosophila melanogaster MOXD1 homolog 2 (olf413)
Recombinant Drosophila melanogaster MOXD1 homolog 2 (olf413) is a transmembrane protein derived from the common fruit fly (Drosophila melanogaster) . This protein is characterized as a homolog of the mammalian MOXD1 (monooxygenase, DBH-like 1) protein, suggesting functional similarities while maintaining species-specific characteristics. In scientific literature and commercial catalogs, this protein is identified by several designations including its primary name olf413, along with alternative gene names such as CG12673, CG14461, CG7495, CR33185, and DmelCG12673 . The protein is also sometimes referred to as "isoform C" in certain contexts, indicating potential variant forms of this protein may exist .
The recombinant versions of this protein are particularly valuable for research purposes as they offer controlled production, consistent quality, and specific modifications such as His-tagging that facilitate detection and purification processes. These recombinant forms are produced through various expression systems, with the most common being E. coli-based production methods that yield high quantities of the protein for experimental use . The availability of this protein in recombinant form enables researchers to conduct detailed studies on its structure, function, and potential interactions within biological systems.
Significance in Scientific Research
As a Drosophila protein with homology to mammalian monooxygenases, olf413 represents an important target for comparative studies across species. The fruit fly serves as a well-established model organism in molecular biology and genetics, making proteins like olf413 valuable for understanding evolutionary conservation of protein structures and functions. The recombinant version allows researchers to isolate and study this specific protein without the complications of working with whole organism extracts, providing cleaner experimental conditions for functional and structural analyses.
Amino Acid Composition and Sequence
The full-length Recombinant Drosophila melanogaster MOXD1 homolog 2 comprises 760 amino acids (positions 1-760) . The complete amino acid sequence, as provided in product specifications, begins with MAHPRKAVATPATLQLGPPA and continues through a series of hydrophilic and hydrophobic regions consistent with its transmembrane nature . This sequence information is critical for researchers performing sequence alignments, structural predictions, or designing experiments that target specific domains within the protein.
The following table summarizes the key structural characteristics of the recombinant protein:
| Characteristic | Specification |
|---|---|
| Protein Length | Full Length (1-760 amino acids) |
| UniProt ID | Q6NP60 |
| Protein Type | Transmembrane Protein |
| Species Origin | Drosophila melanogaster (Fruit fly) |
| Common Tags | N-terminal His-tag, N-terminal 10xHis-tag |
Post-translational Modifications and Tagging
In its recombinant form, the protein is typically modified with an N-terminal histidine tag (His-tag), which facilitates purification using affinity chromatography methods . Some commercial preparations offer a more substantial 10xHis-tag for enhanced binding during purification processes . These tags generally do not interfere with most functional studies and provide a convenient means of detecting the recombinant protein using anti-His antibodies during experimental procedures.
Expression Systems
Recombinant Drosophila melanogaster MOXD1 homolog 2 is produced using various expression systems, with each offering distinct advantages depending on the intended application. The primary expression systems include:
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E. coli Expression: The most commonly used system, providing high yields of the recombinant protein. This prokaryotic expression system is cost-effective but may lack some post-translational modifications present in the native protein .
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Cell-Free Expression: This system allows for the synthesis of proteins without intact cells, potentially offering advantages for proteins that might be toxic to host cells or require specific cofactors for proper folding .
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Alternative Systems: Some manufacturers also offer the protein expressed in yeast, baculovirus, or mammalian cell systems, which may provide more authentic post-translational modifications for specialized applications .
Purity and Quality Control
Commercial preparations of the recombinant protein typically achieve high purity levels, with specifications indicating greater than 90% purity as determined by SDS-PAGE analysis in some products , while others guarantee at least 85% purity . This high level of purity ensures that experimental results are not confounded by the presence of contaminant proteins or degradation products.
The following table summarizes the expression systems and associated purity levels:
| Expression System | Typical Purity | Advantages |
|---|---|---|
| E. coli | >90% | High yield, cost-effective |
| Cell-Free | ≥85% | Good for toxic proteins |
| Yeast/Baculovirus/Mammalian | ≥85% | Better post-translational modifications |
Potential Research Applications
While specific applications are not explicitly detailed in the available search results, the recombinant Drosophila melanogaster MOXD1 homolog 2 would typically be utilized in various research contexts based on its nature as a transmembrane protein with homology to monooxygenases. Potential applications include:
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Structural Studies: Determination of three-dimensional protein structure through techniques such as X-ray crystallography or cryo-electron microscopy.
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Functional Characterization: Investigation of enzymatic activities, particularly those related to monooxygenase functions.
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Protein-Protein Interaction Studies: Identification of binding partners and molecular complexes.
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Comparative Biology: Exploration of evolutionary relationships between Drosophila MOXD1 homolog and related proteins in other species.
Experimental Considerations
When working with this recombinant protein, researchers should consider several factors to optimize experimental outcomes:
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Buffer Compatibility: The protein is formulated in a specific buffer (Tris/PBS with trehalose, pH 8.0), which may need to be considered when designing experiments to avoid buffer incompatibilities.
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Tag Interference: The presence of the His-tag may potentially affect certain protein interactions or enzymatic activities, necessitating control experiments with tag-cleaved versions in some applications.
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Storage Impact: Multiple freeze-thaw cycles should be avoided as they can lead to protein degradation and loss of activity .
Antibodies and Detection Tools
For researchers working with Drosophila melanogaster MOXD1 homolog 2, several related products are commercially available to facilitate detection and characterization. These include:
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Polyclonal Antibodies: Rabbit anti-Drosophila melanogaster olf413 polyclonal antibodies are available for applications such as ELISA and Western Blot analyses . These antibodies are produced through antigen-affinity purification methods and have the IgG isotype.
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Partial Recombinant Proteins: In addition to full-length protein, partial recombinant versions are available that may be useful for mapping specific domains or epitopes .
The following table summarizes the available related products:
| Product Type | Host/Source | Applications | Features |
|---|---|---|---|
| Full-length Recombinant Protein | E. coli | Various research applications | His-tagged, >90% purity |
| Partial Recombinant Protein | Various expression systems | Domain-specific studies | His-tagged, ≥85% purity |
| Polyclonal Antibody | Rabbit | ELISA, Western Blot | IgG isotype, antigen-affinity purified |
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it when placing your order, and we will fulfill your request.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please communicate with us in advance, as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What is MOXD1 homolog 2 (olf413) in Drosophila melanogaster and what are its key characteristics?
Olf413 (UniProt: Q6NP60) is a transmembrane protein in Drosophila melanogaster that functions as a homolog to human MOXD1 (Monooxygenase DBH-like 1). The full-length protein consists of 760 amino acids and belongs to the copper-dependent monooxygenase family . It possesses dopamine beta-monooxygenase activity, copper ion binding properties, and oxidoreductase activity . The protein is involved in several biological processes including:
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Dopamine catabolic process
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Oxidation-reduction processes
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Octopamine biosynthetic process
The protein contains structural features that allow it to function as an enzyme within the monooxygenase pathway, similar to dopamine beta-hydroxylase (DBH) in humans .
What are the recommended storage and handling conditions for recombinant olf413 protein preparations?
For optimal stability and activity of recombinant Drosophila melanogaster MOXD1 homolog 2 (olf413), researchers should follow these evidence-based protocols:
| Parameter | Recommendation | Notes |
|---|---|---|
| Storage temperature | -20°C to -80°C | For extended storage, -80°C is preferred |
| Working aliquots | 4°C | Use within one week |
| Freeze-thaw cycles | Minimize | Repeated freezing and thawing is not recommended |
| Reconstitution | Deionized sterile water | Concentration: 0.1-1.0 mg/mL |
| Glycerol content | 5-50% (final concentration) | 50% is standard for long-term storage |
| Buffer composition | Tris/PBS-based buffer, pH 8.0 | Often contains 6% Trehalose for stability |
Before opening, it is recommended to briefly centrifuge the vial to bring contents to the bottom. The shelf life of the lyophilized form is typically 12 months at -20°C/-80°C, while reconstituted liquid forms have a shelf life of approximately 6 months .
How does the expression pattern of olf413 in Drosophila compare with MOXD1 expression in vertebrate models?
The expression patterns of olf413 in Drosophila and MOXD1 in vertebrates show interesting parallels and differences:
Research has shown that MOXD1 expression is highly conserved between humans and multiple translational models including chickens, mice, and zebrafish . In vertebrates, MOXD1 is particularly important in neural crest-derived tissues, with expression being restricted to mesenchymal cell populations during development .
Similar to vertebrate MOXD1, Drosophila olf413 shows tissue-specific expression patterns relevant to neural function. When designing experiments for comparative studies, researchers should consider that olf413 may show sexual dimorphism in expression levels, as noted for many metabolism-associated genes in Drosophila .
What experimental approaches are most effective for studying olf413 function in Drosophila models?
Based on published methodologies, the following research approaches have proven effective for investigating olf413 function:
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Gene Expression Analysis:
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qRT-PCR using validated reference genes like LamCa, βTub60D and βTub97EF that demonstrate stable expression between sexes
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RNA sequencing to identify olf413-dependent gene networks
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Differential detection (DD) analysis to identify changes in the fraction of cells expressing olf413 across different experimental conditions
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Protein Function Studies:
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Enzymatic activity assays measuring dopamine beta-monooxygenase activity
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Copper binding assays to investigate the role of olf413 in copper metabolism
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Protein interaction studies using co-immunoprecipitation to identify binding partners
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Genetic Manipulation:
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CRISPR-Cas9 mediated knockout strategies to assess loss-of-function phenotypes
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Transgenic overexpression to identify gain-of-function effects
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RNAi knockdown for tissue-specific or temporal silencing
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Developmental Studies:
When selecting reference genes for qRT-PCR analysis, researchers should be aware that common reference genes like GAPDH, β-actin, and 18SrRNA show prominent sexual dimorphism. Instead, structural genes like LamCa and βtub97EF have demonstrated stable expression between sexes and under different nutritional conditions .
What methodological considerations are important when designing CRISPR-Cas9 knockout experiments targeting olf413 in Drosophila?
When designing CRISPR-Cas9 knockout experiments for olf413 in Drosophila, researchers should consider the following critical factors:
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Guide RNA Design and Validation:
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Target early exons to maximize disruption probability
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Sequence verification of mutations is essential to confirm knockout efficiency
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Tissue-Specific Considerations:
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Use appropriate Gal4 drivers for tissue-specific expression of Cas9
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Phenotypic Analysis Methodologies:
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Control Selection:
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Include both wild-type controls and CRISPR controls targeting non-functional regions
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For developmental studies, littermate controls are critical to minimize variability
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Quantification of Knockout Effects:
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Embryonic development can be determined by counting somites or staging
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RNA extraction should use specialized kits for small tissue samples (e.g., RNAqueous Micro Kit)
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How can olf413 research in Drosophila inform our understanding of MOXD1's dual role in tumor biology across different cancer types?
Drosophila olf413 research provides a valuable model for understanding the complex and seemingly contradictory roles of MOXD1 in human cancer biology:
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Context-Dependent Tumor Effects:
Human MOXD1 demonstrates opposing roles in different tumor types: -
Experimental Design Considerations for Drosophila Models:
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Cancer Type-Specific Modeling:
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Design genetic backgrounds that mimic specific human cancer types
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Incorporate human oncogenes (like MYCN for neuroblastoma models)
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Consider tissue-specific expression patterns
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Functional Assays:
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Proliferation assays (EdU incorporation, PH3 staining)
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Migration/invasion assays
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Cell death/apoptosis quantification
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Combined genetic approaches (e.g., olf413 knockdown in combination with known oncogenes)
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Translating Findings Across Species:
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Key Readouts:
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Gene expression profiles of olf413-manipulated tissues
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Developmental timing effects
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Cell lineage-specific effects
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Non-cell-autonomous effects on surrounding tissues
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Research Applications Table:
| Cancer Biology Aspect | Human MOXD1 Finding | Drosophila olf413 Experimental Approach | Expected Outcome |
|---|---|---|---|
| Tumor Suppression | Loss of MOXD1 in neuroblastoma correlates with worse prognosis | olf413 knockdown in neural tissue + oncogene expression | Enhanced tumor growth |
| EMT and Migration | MOXD1 knockout cells more migratory in neuroblastoma | olf413 RNAi in border cells or other migratory cell types | Altered migration patterns |
| Cell Proliferation | MOXD1 KO tumors show higher proportion of mitotic cells | olf413 KO + cell cycle markers in developing tissues | Increased mitotic index |
| Apoptosis Pathway | MOXD1 knockdown in GBM activates ER-mitochondrial apoptosis | olf413 KO + ER stress markers in Drosophila | Activation of ER stress response |
Using Drosophila models to study olf413 can help resolve the seemingly contradictory roles of MOXD1 in different cancer contexts by providing a simplified genetic background for mechanistic studies.
What approaches should researchers use to study the role of olf413 in copper metabolism and its impact on neural development?
Investigating the relationship between olf413, copper metabolism, and neural development requires multifaceted experimental approaches:
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Copper Binding Analysis:
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Direct Binding Assays:
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Isothermal titration calorimetry (ITC) to measure binding affinity
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Site-directed mutagenesis of predicted copper-binding residues
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Spectroscopic analysis of purified recombinant protein
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Neural Development Assessment:
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Morphological Analysis:
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Tissue architecture examination in olf413 mutants
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Developmental timing metrics (e.g., embryonic development staging)
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Integrated Experimental Design:
| Experimental Approach | Methodology | Readouts | Controls |
|---|---|---|---|
| Copper-dependence of olf413 activity | Enzymatic assays with/without copper chelators | Oxidoreductase activity, dopamine metabolism | Copper-independent enzymes |
| Developmental effects of copper modulation | Dietary copper manipulation in olf413 mutants | Developmental timing, neural phenotypes | Wild-type flies on same diets |
| Copper-related gene networks | RNA-seq of olf413 mutants | Expression of copper homeostasis genes | Reference genes stable under copper conditions |
| Tissue-specific copper content | X-ray fluorescence microscopy | Spatial distribution of copper in tissues | Non-neural tissues |
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Data Analysis Framework:
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Compare copper-related phenotypes between olf413 mutants and controls
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Correlate copper binding capacity with enzymatic activity
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Identify gene expression patterns that connect copper metabolism to neural development
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Test whether copper supplementation can rescue olf413 mutant phenotypes
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This integrated approach allows researchers to establish whether copper metabolism is a mechanistic link between olf413 function and neural development outcomes, potentially informing therapeutic approaches for MOXD1-related disorders in humans.
How can differential detection (DD) analysis be optimized for studying olf413 expression patterns across different cell types in Drosophila?
Differential detection (DD) analysis provides valuable insights beyond traditional differential expression analysis, especially for genes like olf413 that may show cell type-specific expression patterns:
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Methodological Framework:
DD analysis identifies genes for which the average fraction of cells with detectable expression changes between groups . For olf413 studies, this approach can reveal:-
Cell populations where olf413 is selectively expressed
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Changes in olf413 detection patterns during development
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Effects of experimental manipulations on olf413 expression breadth
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Optimization for olf413 Studies:
| Analysis Parameter | Recommendation | Rationale |
|---|---|---|
| Detection threshold | Use data-driven approach (e.g., based on negative controls) | olf413 expression may be low in some cell types |
| Cell type resolution | Perform analysis at highest resolution possible | olf413 may have restricted expression patterns |
| Sample integration | Account for batch effects before DD analysis | Ensures biological rather than technical differences |
| Two-stage testing | Combine DD and differential expression (DE) analysis | Provides orthogonal information about olf413 regulation |
| Control genes | Include genes with known expression patterns | Validates analysis sensitivity |
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Interpretation Considerations:
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A gene detected in more cells doesn't necessarily have higher average expression
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Changes in detection can reflect altered cell states or compositions
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For olf413, changes in detection across cell types may indicate functional specialization
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Visualization Strategy:
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Detection rate plots across cell types
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Faceted visualizations by experimental condition
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Combined DD and DE plots to distinguish expression level vs. expression breadth changes
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This approach is particularly valuable for olf413 research as MOXD1 homologs show lineage-restricted expression patterns across species, with important functional implications for development and disease .
