Recombinant Mouse Transmembrane protein C10orf57 homolog (D14Ertd449e)

What is D14Ertd449e and how does it relate to human homologs?

D14Ertd449e (DNA segment, Chr 14, ERATO Doi 449, expressed) is the mouse homolog of human C10orf57 (chromosome 10 open reading frame 57). Both are multi-pass membrane proteins containing 123 amino acids with conserved structural features . The gene encoding D14Ertd449e maps to mouse chromosome 14 A3, while its human counterpart C10orf57 maps to human chromosome 10 . This chromosomal region in humans contains approximately 1,200 genes and constitutes about 4.5% of total cellular DNA, encoding critical proteins including chemokines, cadherins, and excision repair proteins .

The evolutionary conservation of this protein between mouse and human systems makes it a valuable target for comparative studies examining membrane protein function across mammalian species. Understanding this homology relationship is essential when extrapolating experimental findings from mouse models to human applications.

What structural features characterize the D14Ertd449e protein?

D14Ertd449e is classified as a multi-pass membrane protein, indicating it contains multiple transmembrane domains that traverse the lipid bilayer several times . While detailed crystallographic or cryo-EM structural data is not presented in the provided sources, sequence analysis suggests the presence of multiple hydrophobic regions that likely form alpha-helical transmembrane segments.

The protein's 123-amino acid sequence contains characteristic patterns of hydrophobic and hydrophilic residues that are consistent with integral membrane protein architecture . The transmembrane domains are likely connected by both intracellular and extracellular loops, which may be involved in protein-protein interactions or signaling functions. Further structural characterization through techniques such as circular dichroism, limited proteolysis, or computational modeling would provide additional insights into the three-dimensional organization of this membrane protein.

What are the optimal conditions for reconstituting recombinant D14Ertd449e protein?

For optimal reconstitution of lyophilized recombinant D14Ertd449e protein, researchers should first briefly centrifuge the vial to ensure all contents are at the bottom. The protein should be reconstituted in deionized sterile water to a concentration between 0.1-1.0 mg/mL . To enhance stability for long-term storage, it is recommended to add glycerol to a final concentration of 5-50%, with 50% being the standard concentration used by suppliers .

After reconstitution, it is critical to aliquot the protein solution to minimize freeze-thaw cycles, as repeated freezing and thawing can significantly compromise protein integrity and biological activity . For storage, maintain aliquots at -20°C or preferably -80°C for extended periods. Working aliquots may be stored at 4°C but should be used within one week to preserve optimal activity . The reconstitution buffer typically contains Tris/PBS with 6% trehalose at pH 8.0, which helps maintain protein stability .

How can researchers effectively knock down D14Ertd449e expression in mouse models?

For targeted knockdown of D14Ertd449e expression in mouse models or cell lines, researchers have several methodological options. Small interfering RNA (siRNA) provides a direct approach for transient gene silencing. Commercial D14Ertd449e siRNA products typically contain 3.3 nmol of lyophilized siRNA per vial, which yields a 10 μM solution when reconstituted according to manufacturer protocols . This amount is generally sufficient for 50-100 transfections, depending on the experimental setup.

For reconstitution of lyophilized siRNA, researchers should use RNase-free water to create a 10 μM solution in a buffer containing 10 μM Tris-HCl (pH 8.0), 20 mM NaCl, and 1 mM EDTA . Storage of reconstituted siRNA should be at -20°C, with precautions taken to avoid RNase contamination and repeated freeze-thaw cycles that could degrade the RNA molecules.

For longer-term gene silencing, alternative approaches include the use of shRNA plasmids or lentiviral particles targeting D14Ertd449e . These systems provide more sustained knockdown and can be particularly valuable for in vivo studies or experiments requiring extended observation periods. Validation of knockdown efficiency should be performed using qRT-PCR and western blot analysis to confirm reduction in both mRNA and protein levels.

What approaches can be used to study D14Ertd449e localization in cells?

Studying the subcellular localization of D14Ertd449e requires specialized techniques appropriate for membrane proteins. Immunofluorescence microscopy using antibodies against either native D14Ertd449e or epitope tags (such as the His-tag in recombinant versions) can reveal the distribution pattern within cellular compartments . When using tagged recombinant proteins, researchers should verify that the tag does not interfere with proper membrane insertion or trafficking.

For higher resolution analysis, confocal or super-resolution microscopy techniques can be employed to precisely determine the protein's localization within membrane structures. Co-localization studies with markers for specific organelles (endoplasmic reticulum, Golgi apparatus, plasma membrane) can further elucidate the protein's trafficking pathway and resident compartments.

Biochemical fractionation represents another valuable approach, wherein cellular components are separated by differential centrifugation, and D14Ertd449e presence in each fraction is determined by western blot. For definitive localization studies, electron microscopy with immunogold labeling provides nanometer-scale resolution of protein distribution within membrane structures.

How should researchers design experiments to investigate D14Ertd449e function across different mouse strains?

When investigating D14Ertd449e function across different mouse strains, researchers should implement a comprehensive experimental design that accounts for genetic background effects. Based on experimental approaches used for similar comparative studies, a robust design would include multiple inbred strains such as C57BL/6, 129X1/Sv, BALB/c, FVB/N, and DBA/2 . Using at least 12 mice from each strain provides sufficient statistical power to detect strain-specific differences in expression or function .

Tissue collection protocols should be standardized across all strains, with particular attention to the organs where D14Ertd449e is predominantly expressed. RNA-seq or microarray analysis can identify strain-specific differences in expression levels, while proteomics approaches can reveal variations in protein abundance and post-translational modifications. For functional studies, ex vivo tissue preparations or primary cell cultures derived from different strains allow for controlled comparative analyses.

The experimental design should incorporate appropriate controls and account for variables such as age, sex, environmental conditions, and tissue-specific expression patterns. A systematic approach comparing phenotypes across multiple strains can reveal how genetic background influences D14Ertd449e function and potentially identify modifier genes that interact with D14Ertd449e.

What methodological approaches can resolve contradictory data regarding D14Ertd449e membrane topology?

Glycosylation mapping represents a powerful approach wherein potential N-glycosylation sites are introduced at various positions throughout the protein sequence. Since glycosylation occurs only on extracellular or luminal domains, determining which sites become glycosylated can reveal the orientation of different segments of the protein. Similarly, cysteine accessibility assays using membrane-impermeable thiol-reactive reagents can distinguish between cytoplasmic and extracellular cysteine residues.

Protease protection assays provide another methodology, wherein intact membrane preparations are treated with proteases that can only access exposed protein regions. Mass spectrometry analysis of the protected fragments can then reveal which domains were shielded within the membrane or cytoplasmic compartment. For definitive topology mapping, cryo-electron microscopy or X-ray crystallography can provide detailed structural information, though these techniques present significant challenges for membrane proteins like D14Ertd449e.

When confronted with contradictory data, researchers should systematically evaluate the methodological differences between studies, including expression systems, protein tagging strategies, and analytical techniques, which may account for the discrepancies observed.

How can researchers effectively study the relationship between D14Ertd449e and genomic disorders on chromosome 10q?

Investigating the relationship between D14Ertd449e (whose human homolog C10orf57 is located on chromosome 10) and genomic disorders affecting the 10q region requires integrated genomic and functional approaches. Researchers should begin by examining the precise genomic location of C10orf57 relative to known chromosomal deletion or duplication hotspots on chromosome 10q .

Low-copy repeats (LCRs) on chromosome 10q can affect chromosomal stability and are associated with disease-related rearrangements . High-resolution genomic analyses using array-based comparative genomic hybridization (array CGH) or next-generation sequencing can accurately map deletion/duplication breakpoints and determine whether C10orf57 is affected in these genomic disorders . Analysis using SNP arrays can further reveal patterns of loss of heterozygosity (LOH) that might implicate this region .

To establish functional connections, researchers should examine expression levels and protein function of C10orf57/D14Ertd449e in patient-derived cells with 10q abnormalities compared to controls. FISH analysis can confirm the presence or absence of the gene in cases where genomic rearrangements are suspected . For mechanistic studies, CRISPR-Cas9 genome editing can be employed to create cellular or animal models that recapitulate specific 10q deletions or duplications, allowing for direct assessment of phenotypic consequences.

What data table formats are recommended for reporting D14Ertd449e experimental results in grant applications?

When preparing data tables for reporting D14Ertd449e experimental results in grant applications, researchers should adhere to the current FORMS-I data table format required by funding agencies like NIH . These standardized formats ensure consistency and completeness in data presentation, facilitating peer review and cross-study comparisons.

For gene expression studies, data tables should include:

For protein characterization studies, recommended table formats include:

All tables should include detailed footnotes explaining experimental conditions, statistical methods, and any special considerations. When reporting null or negative results, these should be clearly indicated rather than omitted, as they provide valuable information about experimental conditions where effects were not observed.

How should researchers analyze and interpret contradictory data regarding D14Ertd449e function?

When confronted with contradictory data regarding D14Ertd449e function, researchers should implement a systematic analytical framework. Begin by categorizing contradictions as either methodological (arising from different experimental approaches) or biological (reflecting true contextual differences in protein function).

For methodological contradictions, create a detailed comparison table:

This systematic comparison can often reveal that contradictions stem from specific methodological choices rather than fundamental disagreements about protein function. For example, the use of E. coli for expressing a mammalian membrane protein may result in different folding or post-translational modifications compared to mammalian expression systems .

Statistical meta-analysis techniques should be applied when sufficient quantitative data are available across multiple studies. For biological contradictions, consider tissue-specific, developmental, or context-dependent regulation of D14Ertd449e. Design experiments that directly test competing hypotheses under identical conditions, focusing on variables that differ between contradictory reports.

When reporting your analysis, present all relevant data transparently, including contradictory findings, and propose mechanistic explanations that could reconcile opposing results. This approach not only advances scientific understanding but also identifies critical variables affecting D14Ertd449e function.

What bioinformatic approaches are most effective for analyzing D14Ertd449e in genomic datasets?

For effective bioinformatic analysis of D14Ertd449e in genomic datasets, researchers should implement a multi-tiered approach combining sequence-based, structure-based, and network-based methodologies. Begin with comprehensive sequence analysis using tools like BLAST to identify homologs across species, which provides evolutionary context and highlights conserved functional domains .

Multiple sequence alignment using CLUSTAL Omega or similar tools can reveal conserved residues likely critical for protein function. For transmembrane topology prediction, specialized algorithms like TMHMM, HMMTOP, or Phobius should be used, as general prediction tools often perform poorly on multi-pass membrane proteins like D14Ertd449e .

For structural analysis, in the absence of experimental structures, homology modeling using software like MODELLER or I-TASSER can generate predictive three-dimensional models based on structurally characterized homologs. These models should be validated using energy minimization and Ramachandran plot analysis before being used to inform hypotheses about protein function.

Expression correlation networks can identify genes whose expression patterns correlate with D14Ertd449e across tissues or experimental conditions, potentially revealing functional associations. Tools like GeneMANIA or STRING can integrate these correlations with known protein-protein interactions, pathway memberships, and co-expression data.

For analyzing D14Ertd449e in the context of genomic disorders, tools that specialize in identifying structural variations, such as breakpoint mapping algorithms and copy number variation (CNV) detectors, are particularly valuable . Integration of results from multiple bioinformatic approaches provides the most robust framework for generating testable hypotheses about D14Ertd449e function.

How can researchers overcome solubility issues when working with recombinant D14Ertd449e?

Membrane proteins like D14Ertd449e frequently present solubility challenges that can complicate experimental procedures. To overcome these issues, researchers should first optimize the reconstitution process by carefully controlling buffer conditions. The recommended reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL provides a starting point, but further optimization may be necessary .

For enhanced solubility, consider a stepwise approach beginning with the addition of non-ionic detergents such as n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG) at concentrations slightly above their critical micelle concentration (CMC). These detergents can solubilize membrane proteins while maintaining native-like conformations. Alternative solubilization strategies include:

• Using lipid nanodiscs, which provide a more native-like membrane environment

• Employing amphipols, which are amphipathic polymers that wrap around the hydrophobic regions of membrane proteins

• Testing different pH conditions (typically pH 7.0-8.5) and ionic strengths

• Adding glycerol (5-10%) as a stabilizing agent beyond its use as a cryoprotectant

When expressing recombinant D14Ertd449e, consider modifying the construct design to include solubility-enhancing fusion partners such as MBP (maltose-binding protein) or SUMO (small ubiquitin-like modifier). If working with E. coli-expressed protein, be aware that the lack of eukaryotic post-translational modifications might affect solubility compared to protein expressed in mammalian systems .

For analytical techniques requiring completely solubilized protein, more aggressive solubilization with SDS or other ionic detergents may be necessary, though these conditions typically denature the protein and are unsuitable for functional studies.

What strategies can address non-specific binding when studying D14Ertd449e interactions?

Non-specific binding presents a significant challenge when investigating protein-protein interactions involving D14Ertd449e. To minimize this issue, researchers should implement multiple control experiments and optimization strategies. When performing pull-down assays with His-tagged recombinant D14Ertd449e, include appropriate negative controls such as unrelated His-tagged proteins and beads-only conditions to identify background binding .

Optimization strategies to reduce non-specific interactions include:

• Adjusting buffer conditions by increasing salt concentration (typically 150-500 mM NaCl) to disrupt electrostatic interactions

• Adding low concentrations of non-ionic detergents (0.01-0.1% Triton X-100 or NP-40) to reduce hydrophobic interactions

• Including competing proteins such as BSA (0.1-1%) or casein in binding buffers

• Implementing more stringent washing protocols with progressively increasing stringency

• Using cross-linking approaches with defined spacer lengths to capture only directly interacting proteins

When seeking to identify genuine interaction partners, employ multiple complementary techniques such as co-immunoprecipitation, proximity labeling methods (BioID or APEX), and yeast two-hybrid screens. Validation of potential interactions should include reciprocal pull-downs, co-localization studies, and functional assays to confirm biological relevance.

For membrane proteins like D14Ertd449e, special consideration should be given to the membrane environment. Interactions may be detergent-sensitive or dependent on specific lipid compositions, necessitating careful optimization of solubilization conditions or the use of membrane-mimetic systems like nanodiscs or liposomes for interaction studies.