C16orf62 Antibody

What is C16orf62 and why is it relevant to current research?

C16orf62 (Chromosome 16 open reading frame 62) is a protein component of the retriever complex that plays a crucial role in regulating retrograde transport of proteins from endosomes to the trans-Golgi network (TGN) or plasma membrane. The protein has gained significant research interest following its identification as a host dependency factor for porcine deltacoronavirus (PDCoV) infection . Functionally, C16orf62 appears to be essential for the maintenance of certain membrane proteins, particularly aminopeptidase N (APN), which serves as a receptor for several coronaviruses. The protein's involvement in virus entry mechanisms and potential associations with esophageal cancer make it a valuable target for both virology and cancer research .

What types of C16orf62 antibodies are currently available for research applications?

Current research primarily utilizes rabbit polyclonal antibodies raised against recombinant C16orf62. These antibodies are available from multiple suppliers and have been validated for several experimental applications:

These antibodies recognize human C16orf62 (UniProt: Q7Z3J2) and typically target sequences within the N-terminal region .

What are the alternative names for C16orf62 that researchers should be aware of?

When searching literature and databases, researchers should be aware of several alternative designations for C16orf62:

• DKFZp313M0539

• DKFZp434B0212

• FLJ21040

• MGC16824

• Esophageal cancer-associated protein

Using these alternative identifiers in literature searches ensures comprehensive coverage of relevant research .

How should researchers optimize immunohistochemistry protocols for C16orf62 detection?

For optimal immunohistochemistry results with C16orf62 antibodies, researchers should:

• Use paraformaldehyde (PFA) fixed samples, as this fixation method preserves C16orf62 epitopes effectively.

• Begin with a dilution range of 1:200-1:500 for polyclonal antibodies and optimize for specific tissue types.

• Include positive control tissues with known C16orf62 expression patterns.

• Validate antibody specificity using tissues or cells with confirmed C16orf62 knockout or knockdown.

• For antigen retrieval, use citrate buffer (pH 6.0) with heat-induced epitope retrieval methods.

The optimal working dilution should be determined experimentally for each specific application and tissue type, as performance may vary across different sample preparations .

What are the validated applications for currently available C16orf62 antibodies?

Based on the available research data, C16orf62 antibodies have been validated for:

• Western blotting: Detecting C16orf62 protein (expected molecular weight approximately 124 kDa)

• Immunohistochemistry: With both paraffin-embedded and PFA-fixed specimens

• Protein-protein interaction studies: Including co-immunoprecipitation experiments to detect interactions with other proteins such as APN

These applications have been successfully employed to study C16orf62's role in viral infection mechanisms and retrograde transport pathways .

What controls should be included when using C16orf62 antibodies in experimental procedures?

To ensure reliable and interpretable results, researchers should implement the following controls when using C16orf62 antibodies:

• Positive tissue/cell controls: Samples with confirmed C16orf62 expression

• Negative controls:

  • Primary antibody omission

  • Isotype controls (rabbit IgG at equivalent concentration)

  • C16orf62 knockout cell lines (especially important for specificity validation)

• Loading controls for western blots: Standard housekeeping proteins such as β-actin or GAPDH

• Validation using multiple antibody clones or detection methods when possible

The genome-wide CRISPR/Cas9 screening studies provide valuable resources for generating C16orf62 knockout cell lines that can serve as effective negative controls .

How can C16orf62 antibodies be utilized to investigate viral infection mechanisms?

C16orf62 antibodies provide valuable tools for investigating viral infection mechanisms, particularly for coronaviruses that utilize APN as a receptor:

• Co-localization studies: Using confocal microscopy with C16orf62 and viral protein antibodies to visualize interactions during infection

• Protein complex detection: Through co-immunoprecipitation to identify virus-host protein interactions

• Cell surface expression analysis: Flow cytometry or immunofluorescence to measure how C16orf62 affects receptor expression

• Infection progression visualization: Tracking the role of C16orf62 during different stages of viral infection

Research has demonstrated that C16orf62 is essential for PDCoV attachment and internalization, with knockout cells showing significantly reduced viral binding and internalization. Antibodies against C16orf62 can help visualize its colocalization with APN and viral components during infection .

What methodological approaches can be used to study C16orf62's role in the retriever complex?

To investigate C16orf62's function within the retriever complex, researchers can employ several antibody-dependent approaches:

• Proximity ligation assays: To detect in situ interactions between C16orf62 and other retriever complex components (DSCR3, VPS29)

• Immunoprecipitation followed by mass spectrometry: To identify the complete interactome of C16orf62

• FRET/BRET assays: To study dynamic protein-protein interactions in living cells

• Subcellular fractionation combined with western blotting: To track C16orf62 localization across cellular compartments

• Immunofluorescence time-course experiments: To visualize trafficking events mediated by the retriever complex

These approaches have revealed that C16orf62 interacts with cargo adaptors like SNX17 to facilitate protein recycling from endosomes to the plasma membrane, a process crucial for maintaining proper receptor expression levels .

How can researchers investigate the relationship between C16orf62 and receptor expression levels?

The research indicates C16orf62 plays a critical role in maintaining cell surface expression of certain receptors, particularly APN. To further investigate this relationship, researchers can:

• Perform quantitative western blot analysis comparing receptor levels in wild-type versus C16orf62 knockout cells

• Use cell surface biotinylation assays to specifically quantify membrane-localized receptors

• Employ pulse-chase experiments with receptor antibodies to track internalization and recycling rates

• Conduct flow cytometry with surface-specific antibodies to measure receptor expression in live cells

• Perform rescue experiments by reintroducing C16orf62 in knockout cells and measuring receptor recovery

These approaches have demonstrated that C16orf62 knockout results in reduced APN expression at the cell surface, with consequent impairment of virus adsorption for APN-dependent viruses .

What are the optimal storage and handling conditions for C16orf62 antibodies?

To maintain antibody functionality and prevent degradation, researchers should:

• Store antibody stocks at 4°C for short-term use (1-2 weeks)

• For long-term storage, maintain at -20°C or preferably -80°C

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles (each cycle can reduce activity)

• Add carrier protein (BSA, 1-5 mg/ml) if diluting for storage

• Avoid exposure to light for fluorophore-conjugated antibodies

• Centrifuge briefly before opening to collect solution at the bottom of the vial

The specific formulation of commercially available C16orf62 antibodies (typically in PBS, pH 7.5 with 40% glycerol and 0.02% sodium azide, or in 0.1M Tris pH 7.0 with 0.1M Glycine and 10% Glycerol) helps maintain stability, but proper handling remains essential .

How should researchers design experiments to investigate C16orf62's role in viral infection?

When designing experiments to study C16orf62's role in viral infection pathways, researchers should consider the following methodological approaches:

• Generate clonal C16orf62 knockout cell lines using CRISPR/Cas9, confirming disruption through sequencing

• Perform virus binding and internalization assays using qRT-PCR to quantify viral particles

• Include rescue experiments with C16orf62 expression vectors to confirm specificity of observed effects

• Utilize fluorescent reporter viruses (such as PDCoV-GFP) to visualize infection in real-time

• Design time-course experiments to determine which stage of the viral life cycle is affected

• Compare effects across multiple susceptible cell lines to ensure reproducibility of findings

Research demonstrates that knockout of C16orf62 significantly reduces PDCoV infection, with effects on both virus binding and internalization stages. Rescue experiments confirm specificity by partially restoring viral infection capability .

What approaches can resolve potential discrepancies in C16orf62 antibody detection results?

When facing inconsistent results with C16orf62 antibodies, researchers should implement these troubleshooting strategies:

• Validate antibody specificity with knockout controls and multiple detection methods

• Test multiple antibody concentrations and incubation conditions

• Use different epitope-targeting antibodies to rule out epitope masking issues

• Verify protein extraction methods are appropriate for membrane-associated proteins

• Consider post-translational modifications that might affect antibody recognition

• For co-localization studies, ensure proper controls for bleed-through and cross-reactivity

When investigating C16orf62's interactions with viral proteins, co-immunoprecipitation experiments revealed that C16orf62 does not directly interact with the PDCoV S1 protein but instead affects receptor availability through its interaction with APN. Such findings highlight the importance of using multiple experimental approaches to resolve seemingly contradictory results .

How can researchers address potential off-target effects when studying C16orf62 function?

When investigating C16orf62 function through gene knockout or knockdown, researchers should implement these strategies to address potential off-target effects:

• Use multiple guide RNAs or siRNAs targeting different regions of C16orf62

• Generate multiple independent knockout clones and compare phenotypes

• Perform rescue experiments by re-expressing C16orf62 in knockout cells

• Check expression of other retriever complex components (DSCR3, VPS29) to ensure specific effects

• Employ targeted gene editing approaches that introduce minimal perturbations to genomic context

• Validate phenotypes using pharmacological inhibitors of pathways when available

Research on C16orf62's role in PDCoV infection employed rescue experiments that partially restored viral infection capability in knockout cells, confirming specificity of the observed phenotype. Additionally, multiple knockout cell lines showed consistent resistance to infection, supporting a genuine role for C16orf62 in this process .

What experimental design considerations should be made when studying C16orf62's interactions with other proteins?

To effectively investigate C16orf62's protein-protein interactions, researchers should:

• Use multiple interaction detection methods:

  • Co-immunoprecipitation with antibodies against different interacting partners

  • Proximity ligation assays for in situ detection

  • FRET/BRET approaches for live-cell interaction studies

• Include appropriate controls:

  • Negative controls with unrelated proteins of similar size/localization

  • Positive controls with known interaction partners

  • Input controls to confirm protein expression

• Consider interaction dynamics:

  • Test interactions under different cellular conditions

  • Investigate effects of mutations in key domains

  • Examine temporal aspects of interactions

Research has demonstrated that C16orf62 interacts with APN through co-immunoprecipitation experiments and confocal microscopy showing colocalization. These interactions appear functionally significant, as C16orf62 knockout reduces APN expression at the cell surface .

What are promising research applications for C16orf62 antibodies beyond viral infection studies?

Given C16orf62's role in membrane protein trafficking and its association with esophageal cancer, several promising research directions emerge:

• Cancer biology: Investigating C16orf62 expression patterns in different cancer types and correlation with disease progression

• Membrane trafficking: Exploring its role in maintaining expression of other clinically relevant receptors

• Neurodegenerative diseases: Examining potential involvement in protein recycling pathways relevant to neurodegeneration

• Drug development: Using C16orf62 antibodies to screen for compounds that modulate retriever complex function

• Developmental biology: Studying its role in tissue-specific receptor expression during development

The identification of C16orf62 as both a potential cancer-associated protein and a critical host factor for viral infection suggests it may serve as an important nexus in multiple cellular pathways worthy of further investigation .

How might researchers utilize C16orf62 antibodies to develop novel antiviral approaches?

The essential role of C16orf62 in facilitating coronavirus infection suggests several potential therapeutic strategies:

• Screening for small molecules that interfere with C16orf62 function or its interactions with trafficking machinery

• Developing cell-permeable peptides that disrupt specific protein-protein interactions involving C16orf62

• Using C16orf62 antibodies to identify critical functional domains that could be targeted by inhibitors

• Investigating combination approaches targeting both viral proteins and host dependency factors like C16orf62

• Exploring whether modulation of C16orf62 function affects infection by other viruses that utilize similar entry mechanisms

Research has demonstrated that C16orf62 knockout significantly reduces PDCoV infection, and the same effect was observed for TGEV infection, suggesting a broader role in coronavirus entry that could be leveraged for therapeutic development .

What methodological advances would enhance C16orf62 research capabilities?

To advance C16orf62 research, several methodological developments would be valuable:

• Generation of monoclonal antibodies targeting different domains of C16orf62

• Development of proximity-labeling approaches to identify the complete C16orf62 interactome in different cellular contexts

• Creation of inducible and tissue-specific knockout models to study C16orf62 function in vivo

• Establishment of high-throughput screening platforms to identify modulators of C16orf62 function

• Application of cryo-EM and structural biology approaches to determine the three-dimensional structure of C16orf62 within the retriever complex

These methodological advances would provide researchers with more precise tools to dissect C16orf62's functions and potential as a therapeutic target .