Recombinant Mouse Uncharacterized membrane protein C19orf24 homolog

What is known about the tissue expression pattern of mouse C19orf24 homolog?

While comprehensive expression data for the mouse C19orf24 homolog is limited, inferences can be made from the human ortholog studies. The human C19orf24 demonstrates highly specific expression, being found primarily in liver tissue and showing preferential expression in normal tissue compared to diseased states .

For mouse homolog expression analysis, researchers should consider:

• Conducting tissue-specific RT-PCR across major organ systems

• Performing immunohistochemistry with specific antibodies against the mouse protein

• Analyzing publicly available RNA-seq datasets from mouse tissue panels

A methodical approach would involve validating expression at both mRNA and protein levels, as post-transcriptional regulation may affect actual protein abundance in different tissues.

What are the predicted structural features of mouse C19orf24 homolog?

Based on sequence analysis and comparison with the human ortholog, the mouse C19orf24 homolog likely contains transmembrane domains, consistent with its classification as a membrane protein . The protein sequence (amino acids 21-108) suggests a small protein with potential membrane-spanning regions.

The human ortholog has been shown to lack a conventional signal peptide despite being secreted, indicating a non-classical secretory pathway . Similarly, the mouse homolog may utilize alternative secretion mechanisms. The protein has been confirmed to have no N-glycosylation modification sites based on deglycosylation analysis with PNGase F in the human version , and this characteristic is likely conserved in the mouse homolog.

Predicted structural features include:

• Transmembrane domains

• Absence of conventional signal peptide

• No N-glycosylation sites

• Potential protein-protein interaction motifs (requires experimental validation)

What are recommended experimental applications for recombinant mouse C19orf24 homolog?

The recombinant mouse C19orf24 homolog protein can be utilized in several experimental applications:

• Secretion pathway studies: Since the human ortholog follows a non-classical secretion pathway not dependent on the Golgi apparatus , the mouse protein can be used to investigate evolutionary conservation of this unconventional secretion mechanism.

• Protein-protein interaction studies: Pull-down assays utilizing the His-tagged recombinant protein to identify binding partners in mouse tissue lysates, particularly from liver.

• Antibody production: As a full-length recombinant protein, it serves as an excellent immunogen for developing specific antibodies for immunodetection methods.

• Functional characterization: In vitro assays to determine potential enzymatic activities or signaling capabilities.

• Subcellular localization studies: Fluorescently labeled protein can be used to track localization patterns in mouse cell lines.

How should I design experiments to study the secretion mechanism of mouse C19orf24 homolog?

Based on findings with the human ortholog, a comprehensive approach to studying the secretion mechanism would include:

• Brefeldin A (BFA) treatment assays: The human protein maintains secretion despite BFA treatment, which inhibits classical secretory pathways . Design an experiment where cells expressing mouse C19orf24 are treated with BFA, followed by analysis of culture supernatants for secreted protein.

• Subcellular co-localization studies:

  • Transfect cells with fluorescently tagged mouse C19orf24 homolog

  • Counter-stain with markers for different cellular compartments (ER, Golgi, endosomes)

  • The human ortholog does not co-localize with Golgi markers , so compare localization patterns

• Mutagenesis analysis:

  • Create truncation mutants to identify secretion-essential domains

  • Modify potential post-translational modification sites

  • Analyze the effect on secretion efficiency

• Secretion kinetics:

  • Pulse-chase experiments to track the time course of protein synthesis to secretion

  • Compare with classically secreted proteins as controls

• Inhibitor panel screening:

  • Test various pathway inhibitors beyond BFA (e.g., monensin, bafilomycin A1)

  • This can help narrow down the specific non-classical pathway involved

What control proteins should be used when studying mouse C19orf24 homolog?

When designing experiments involving mouse C19orf24 homolog, appropriate controls are essential:

For secretion studies:

• Positive control: A classical secreted protein with signal peptide (e.g., serum albumin)

• Negative control: A cytosolic protein not expected to be secreted (e.g., GAPDH)

• Comparative control: Another non-classical secreted protein (e.g., FGF1, IL-1β)

For membrane localization studies:

• Known transmembrane proteins with similar topology

• For subcellular fractionation: markers for each cellular compartment

For functional studies:

• Consider related proteins from the same family (other FAM174 proteins)

• Proteins with similar evolutionary patterns (recent evolution, purifying selection)

The specific choice of control proteins should be tailored to the hypothesis being tested and the experimental system being used.

What functional domains have been identified in mouse C19orf24 homolog?

• Transmembrane domain: The sequence "LFYVITGLCGLISLYFLIRAF" within the protein contains hydrophobic residues consistent with a membrane-spanning region .

• Potential interaction motifs: The C-terminal region "TEEHEEMASQDSEEETVFETRNLR" contains multiple glutamic acid residues that could be involved in protein-protein interactions through charged interactions.

Experimental approaches to identify functional domains would include:

• Systematic deletion analysis to map regions essential for localization and secretion

• Yeast two-hybrid or pull-down assays to identify interacting proteins

• Point mutation analysis of conserved residues to identify functionally important amino acids

How can I determine the subcellular localization of mouse C19orf24 homolog?

To determine the subcellular localization of mouse C19orf24 homolog, several complementary approaches should be employed:

• Fluorescence microscopy with tagged protein:

  • Express fluorescently tagged protein (e.g., GFP-fusion) in appropriate mouse cell lines

  • Co-stain with markers for cellular compartments (ER, Golgi, plasma membrane)

  • Perform confocal microscopy for high-resolution localization

• Subcellular fractionation:

  • Homogenize cells/tissues expressing the protein

  • Separate cellular compartments via differential centrifugation

  • Detect the protein in each fraction via Western blotting

  • Compare distribution with known compartment markers

• Immunoelectron microscopy:

  • Provides ultrastructural localization at the nanometer scale

  • Essential for precise membrane localization

• Cell surface biotinylation:

  • To determine if the protein reaches the plasma membrane

  • Compare with total protein levels to estimate trafficking efficiency

Based on studies of human C19orf24, it would be particularly important to examine localization in relation to the Golgi apparatus, as the human protein does not co-localize there despite being secreted , suggesting a novel trafficking pathway.

What approaches should be used to identify binding partners of mouse C19orf24 homolog?

Identifying binding partners is critical for understanding the function of this uncharacterized protein. Several complementary approaches should be considered:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Use His-tagged recombinant mouse C19orf24 homolog as bait

  • Incubate with mouse tissue lysates (focus on liver based on human expression data)

  • Purify complexes using nickel affinity chromatography

  • Identify bound proteins by mass spectrometry

• Proximity labeling approaches:

  • Generate BioID or APEX2 fusion constructs with mouse C19orf24 homolog

  • Express in relevant cell types

  • Activate labeling and purify biotinylated proteins

  • Identify proximal proteins by mass spectrometry

• Yeast two-hybrid screening:

  • Use full-length protein and various domains as bait

  • Screen against mouse cDNA libraries (liver-derived would be most relevant)

• Co-immunoprecipitation validation:

  • Confirm interactions identified by high-throughput methods

  • Use in physiologically relevant cell types

• Functional validation of interactions:

  • Knockdown studies of potential interactors

  • Competition assays with peptides derived from interaction domains

For all approaches, stringent controls are essential to distinguish true interactors from background binding.

What is known about the evolutionary history of C19orf24 and its homologs?

The evolutionary history of C19orf24 is particularly interesting as studies on the human ortholog have revealed it to be a recently evolved gene. Key evolutionary features include:

• Recent emergence: The human C19orf24 gene appears to be found only in humans and chimpanzees (Pan troglodytes), suggesting a very recent evolutionary origin .

• Purifying selection: Analysis of synonymous and non-synonymous substitution rates (dS/dN) for the human gene indicates it has undergone purifying selection . This suggests that despite its recent origin, the gene has acquired an important biological function that is being maintained by evolutionary pressure.

For mouse C19orf24 homolog research, evolutionary analyses should include:

• Comparative genomics across rodent species to determine conservation

• Comparison with primate sequences to identify regions of convergent evolution

• Analysis of selection patterns in mice compared to humans

The recent evolution of this gene family makes it particularly interesting for studying the emergence of novel gene functions in mammals.

How conserved is C19orf24 across species, and what might this tell us about its function?

The limited conservation of C19orf24 across species provides important clues about its function:

• Restricted taxonomic distribution: The human version appears limited to humans and chimpanzees , suggesting a specialized function in higher primates.

• Functional constraints: Despite its recent emergence, the evidence of purifying selection suggests functional constraints are already in place .

For the mouse homolog, researchers should:

• Perform detailed sequence alignments between mouse and human proteins

• Identify absolutely conserved residues as potentially functionally critical

• Examine expression patterns across tissues to identify conservation of regulatory mechanisms

The limited conservation suggests this protein may be involved in species-specific adaptations rather than fundamental cellular processes, which would typically show broader conservation across evolutionary time.

What research approaches can help elucidate the biological significance of this evolutionarily young protein?

To understand the biological significance of this evolutionarily young protein:

• Comparative functional studies:

  • Compare properties of mouse and human orthologs in the same cellular context

  • Test whether functions are conserved across species

• Knockout/knockdown models:

  • Generate mouse knockout models or cell lines with CRISPR-Cas9

  • Analyze phenotypes for clues to biological function

  • Compare with knockdown effects in human cells

• Expression profiling under various conditions:

  • Examine regulation during development

  • Test responses to different physiological stresses

  • Analyze expression in disease models

• Evolutionary rate analysis:

  • Compare evolutionary rates of different protein domains

  • Identify regions under strongest selection pressure

• Ancestral sequence reconstruction:

  • Reconstruct the likely ancestral sequence

  • Test functional differences between ancestral and modern proteins

These approaches can help determine whether this protein represents a functional innovation specific to certain mammalian lineages and what selective advantages it might confer.

What are the optimal storage and handling conditions for recombinant mouse C19orf24 homolog?

For optimal stability and activity of recombinant mouse C19orf24 homolog protein:

Storage conditions:

• Store lyophilized protein at -20°C/-80°C upon receipt

• After reconstitution, store working aliquots at 4°C for up to one week

• For long-term storage, add 5-50% glycerol (50% recommended final concentration) and store at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as this can damage protein structure and activity

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Gently mix by inversion or slow rotation, avoid vigorous shaking

• Prepare single-use aliquots to minimize freeze-thaw cycles

Buffer compatibility:

• The protein is supplied in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• For functional assays, consider buffer exchange if the experimental system requires specific ionic conditions

Quality control considerations:

• Verify protein integrity by SDS-PAGE before use in critical experiments

• Standard product should have >90% purity as determined by SDS-PAGE

What methods are available for validating the activity of recombinant mouse C19orf24 homolog?

Since the specific biological activity of C19orf24 homolog remains largely uncharacterized, a multi-faceted approach to validation is recommended:

• Structural integrity validation:

  • SDS-PAGE to confirm correct molecular weight and purity

  • Circular dichroism (CD) spectroscopy to verify secondary structure

  • Limited proteolysis to assess proper folding

• Functional validation options:

  • Secretion assay: Test if the recombinant protein maintains the ability to be secreted when expressed in cells

  • Localization assay: Verify proper subcellular localization using fluorescently tagged protein

  • Binding assays: If binding partners are identified, test interaction with purified partners

• Comparison with native protein:

  • Western blot comparing recombinant and native proteins from mouse tissues

  • Mass spectrometry validation of post-translational modifications

• Species cross-reactivity testing:

  • Test functional equivalence between mouse and human orthologs in the same assay systems

Without a defined enzymatic activity, these structural and comparative approaches provide the best validation of proper protein production and folding.

What are common technical challenges when working with recombinant mouse C19orf24 homolog?

Researchers working with this protein should be aware of several potential technical challenges:

• Membrane protein solubility issues:

  • As a membrane protein, C19orf24 homolog may have solubility limitations

  • Consider testing different detergents for extraction and stabilization

  • Optimize buffer conditions to maintain native structure

• Non-classical secretion detection:

  • Based on the human ortholog's non-classical secretion , standard secretion assays may need modification

  • Be prepared to detect secreted protein in unexpected cellular fractions

  • Use multiple detection methods (Western blot, ELISA, fluorescent tags)

• Functional assay development:

  • The lack of characterized function makes activity assays challenging

  • Start with binding assays and localization studies

  • Consider developing reporter systems based on localization or secretion

• Antibody specificity concerns:

  • Due to limited characterization, commercial antibodies may have specificity issues

  • Validate antibodies using recombinant protein and knockout/knockdown controls

  • Consider generating custom antibodies using the recombinant protein as immunogen

• Expression system limitations:

  • While typically expressed in E. coli , mammalian expression systems may be needed for certain applications requiring authentic folding and trafficking

  • When using E. coli-expressed protein, confirm proper folding before functional studies

Being aware of these challenges and planning experiments accordingly will help researchers obtain reliable results when working with this relatively uncharacterized protein.

What disease models might benefit from research involving mouse C19orf24 homolog?

Based on the known characteristics of C19orf24, several disease models might benefit from research involving the mouse homolog:

• Liver-specific disorders: Given the human ortholog's specific expression in liver tissue , mouse models of hepatic diseases could be relevant for studying C19orf24 homolog function.

• Secretory pathway disorders: As a protein utilizing a non-classical secretory pathway , C19orf24 research could provide insights into diseases involving secretory pathway dysregulation.

• Evolutionary medicine: Since C19orf24 is a recently evolved gene in primates , studying the mouse homolog could help understand species-specific disease susceptibilities and mechanisms.

Research approaches should include:

• Expression analysis in mouse models of liver disease

• Correlation of expression levels with disease progression

• Knockout/overexpression studies in relevant disease models

How can researchers effectively study potential signaling pathways involving C19orf24 homolog?

To investigate potential signaling pathways involving mouse C19orf24 homolog:

• Phosphoproteomic analysis:

  • Compare global phosphorylation changes in cells overexpressing or lacking C19orf24

  • Identify affected signaling nodes and pathways

• Interactome mapping:

  • Use systems biology approaches to place identified binding partners in known pathways

  • Create network models of potential signaling connections

• Functional genomic screens:

  • Conduct CRISPR screens to identify genetic interactions

  • Look for synthetic lethal or synthetic viable interactions with C19orf24 knockout

• Stimulus-response studies:

  • Monitor C19orf24 expression and localization in response to various stimuli

  • Test whether C19orf24 levels affect cellular responses to these stimuli

• Receptor-ligand interaction testing:

  • Given its secreted nature in humans , test whether the mouse homolog can function as a ligand for cell surface receptors

  • Screen receptor libraries for potential binding interactions

These approaches can help place this uncharacterized protein in a functional context and identify the signaling pathways it may influence.

What collaborative research models would accelerate understanding of C19orf24 homolog function?

Understanding the function of this uncharacterized protein would benefit from interdisciplinary collaboration:

• Structural biology and biochemistry:

  • Determine the three-dimensional structure of the protein

  • Characterize biochemical properties and potential enzymatic activities

• Evolutionary biology and genomics:

  • Analyze sequence evolution across mammalian species

  • Identify patterns of selection and conservation

• Cell biology and imaging:

  • Track protein trafficking and secretion in real-time

  • Map detailed subcellular localization

• Physiology and mouse models:

  • Generate and characterize knockout mice

  • Perform tissue-specific conditional deletion studies

• Computational biology and systems biology:

  • Predict protein function through advanced algorithms

  • Model potential interaction networks

An integrated research approach combining these disciplines would accelerate understanding of this protein's biological role and significance.

What is the recommended experimental workflow for first-time studies of mouse C19orf24 homolog?

For researchers beginning work with mouse C19orf24 homolog, the following stepwise approach is recommended:

• Initial characterization:

  • Verify protein quality by SDS-PAGE and Western blot

  • Confirm size and purity before proceeding to functional studies

  • Check protein stability under experimental conditions

• Expression profiling:

  • Determine tissue expression pattern in mouse (RT-PCR and Western blot)

  • Compare with human expression data (primarily liver-specific)

  • Analyze expression under different physiological conditions

• Subcellular localization:

  • Express tagged protein in relevant cell lines

  • Perform co-localization studies with organelle markers

  • Compare with human ortholog's non-Golgi localization pattern

• Secretion analysis:

  • Test if mouse homolog is secreted like human ortholog

  • Determine sensitivity to secretory pathway inhibitors (BFA)

  • Characterize secretion kinetics and efficiency

• Interaction studies:

  • Identify binding partners through pull-down or proximity labeling

  • Validate key interactions by co-immunoprecipitation

  • Map interaction domains through truncation mutants

This sequential approach builds understanding from basic properties to more complex functional aspects.

How should researchers interpret conflicting data regarding C19orf24 homolog function?

When encountering conflicting data about C19orf24 homolog function:

• Consider species differences:

  • The human ortholog is evolutionarily recent - mouse homolog may have distinct functions

  • Compare experimental conditions across species carefully

• Evaluate experimental conditions:

  • Cell type-specific effects may explain disparate results

  • Different expression levels can lead to varying outcomes

  • Buffer conditions may affect protein behavior, especially for membrane proteins

• Resolve discrepancies methodically:

  • Replicate both conflicting findings using identical protocols

  • Test intermediate conditions to identify threshold effects

  • Consider combinatorial factors that may reconcile differences

• Technical validation:

  • Verify antibody specificity using knockout controls

  • Confirm construct sequences and expression levels

  • Use multiple detection methods for crucial observations

• Context-dependent function:

  • Consider that this protein may have different functions in different cellular contexts

  • Test hypotheses about conditional functionality