C16orf70 Antibody

What is the function of C16orf70 and why is it important for antibody-based research?

C16orf70 (also known as PHAF1) functions as a regulatory protein in autophagic processes. It forms a complex with BCAS3 and associates with autophagosome formation sites during both non-selective and selective autophagy . This protein's involvement in cellular degradation pathways makes it a significant target for researchers studying autophagy mechanisms, neurodegenerative disorders, cancer biology, and cellular stress responses.

When designing experiments with C16orf70 antibodies, researchers should consider the protein's subcellular localization and expression patterns in different tissue types. The antibody-based detection of C16orf70 can provide valuable insights into autophagy regulation mechanisms and potential therapeutic targets for diseases involving dysregulated autophagy. Western blot analysis typically shows a band at approximately 48 kDa, which serves as a validation marker for antibody specificity .

What antibody types are available for C16orf70 detection and what are their applications?

Several types of C16orf70 antibodies are available for research applications, including:

• Mouse polyclonal antibodies - Unconjugated, IgG isotype, with human reactivity

• Rabbit recombinant monoclonal antibodies - Suitable for immunoprecipitation (IP) and western blot (WB)

• Rabbit polyclonal antibodies - Affinity isolated, used for immunohistochemistry (IHC), immunofluorescence (IF), and immunoblotting

These antibodies can be applied in multiple techniques:

• Western blot (WB): Typically used at 0.04-0.4 μg/mL concentration

• Immunoprecipitation (IP): Effectively used at 1/30 dilution in experimental conditions

• Immunohistochemistry (IHC): Recommended dilutions of 1:200-1:500

• Immunofluorescence (IF): Optimal at 0.25-2 μg/mL concentration

The choice of antibody depends on the specific research application, with monoclonal antibodies offering higher specificity and polyclonal antibodies providing broader epitope recognition.

How should researchers validate C16orf70 antibody specificity before experimental use?

Validating antibody specificity is critical for obtaining reliable research results. For C16orf70 antibodies, a multi-step validation approach is recommended:

• Western blot validation: Verify the presence of a single band at the expected molecular weight (approximately 48 kDa) . Compare results across multiple cell lines known to express C16orf70, such as SH-SY5Y, U87-MG, and JAR cells .

• Positive controls: Include lysates from human fetal brain tissue, which shows clear C16orf70 expression .

• Recombinant protein controls: Consider using recombinant C16orf70 protein (such as those with Myc-DYKDDDDK Tag) as a positive control for antibody validation .

• Cross-reactivity testing: Assess potential cross-reactivity against a protein array containing human recombinant protein fragments, as performed by suppliers like Prestige Antibodies .

• Immunohistochemistry verification: Test antibody performance across multiple tissue types to ensure consistent staining patterns across samples where the protein is expected to be expressed.

This validation workflow ensures antibody specificity and minimizes the risk of false positive or negative results in subsequent experiments.

What are the optimal protocols for using C16orf70 antibodies in studying autophagy mechanisms?

When investigating autophagy mechanisms using C16orf70 antibodies, optimized protocols should address the protein's role in autophagosome formation. The following methodological approach is recommended:

Immunofluorescence co-localization studies:

• Fix cells using 4% paraformaldehyde (15 minutes at room temperature)

• Permeabilize with 0.1% Triton X-100 in PBS (10 minutes)

• Block with 5% BSA (1 hour)

• Incubate with primary C16orf70 antibody at 0.25-2 μg/mL concentration overnight at 4°C

• Co-stain with autophagy markers (LC3B, p62/SQSTM1, BECN1) to assess co-localization

• Visualize using confocal microscopy to determine spatial relationships

Co-immunoprecipitation for BCAS3 interaction analysis:

• Prepare cell lysates in non-denaturing buffer containing protease inhibitors

• Pre-clear lysates with protein A/G beads

• Incubate cleared lysates with C16orf70 antibody (1/30 dilution as recommended for IP)

• Precipitate complexes with protein A/G beads

• Wash extensively and elute

• Analyze by western blotting for both C16orf70 (48 kDa) and BCAS3

For autophagy flux assessment, researchers should combine C16orf70 antibody staining with treatments using autophagy modulators (bafilomycin A1, rapamycin) to determine how C16orf70 localization and expression change during autophagy induction or inhibition.

How can researchers troubleshoot non-specific binding or weak signals when using C16orf70 antibodies in immunoblotting?

Non-specific binding and weak signals are common challenges when working with C16orf70 antibodies. The following troubleshooting methodology addresses these issues:

For non-specific binding:

• Optimize blocking conditions: Test different blocking agents (5% BSA, 5% non-fat milk, commercial blocking buffers) to reduce background. For C16orf70 detection, 5% BSA in TBST often provides superior results.

• Titrate antibody concentration: While manufacturers recommend 0.04-0.4 μg/mL for immunoblotting , validation experiments suggest that 0.2 μg/mL provides optimal signal-to-noise ratio for most cell types.

• Increase washing stringency: Use TBST with 0.1% Tween-20 and perform 5-6 washes of 5-10 minutes each after both primary and secondary antibody incubations.

• Add competing peptides: If specific bands are difficult to distinguish, use the immunogen sequences provided by manufacturers (such as "NQRLKVIEV CDLTKVKLKY CGVHFNSQAI APTIEQIDQS FGATHPGVYN SAEQLFHLNF RGLSFSFQLD SWTEAPKYEP NFAHGLASLQ IPHGATVKRM Y") to pre-absorb the antibody.

For weak signals:

• Sample preparation optimization: Include phosphatase and protease inhibitors in lysis buffers to prevent degradation of C16orf70.

• Increase protein loading: Load 25-30 μg of total protein instead of the standard 20 μg used in published protocols .

• Enhance detection sensitivity: Use enhanced chemiluminescence (ECL) substrates with higher sensitivity or consider fluorescent secondary antibodies for quantitative analysis.

• Extended antibody incubation: Increase primary antibody incubation to overnight at 4°C and secondary antibody to 2 hours at room temperature.

Validation experiments show that these adjustments significantly improve detection of the 48 kDa C16orf70 band across multiple cell lines.

What experimental strategies can be employed to study the interaction between C16orf70 and BCAS3 in autophagosome formation?

Investigating the C16orf70-BCAS3 interaction in autophagosome formation requires sophisticated experimental approaches:

Proximity Ligation Assay (PLA):

• Fix cells using 4% paraformaldehyde

• Permeabilize with 0.1% Triton X-100

• Block with 5% BSA

• Incubate with primary antibodies: rabbit anti-C16orf70 (1:200) and mouse anti-BCAS3

• Perform PLA according to manufacturer's protocol

• Counterstain for autophagosome markers

• Quantify PLA signals in relation to autophagosome structures

CRISPR/Cas9-mediated tagging:

• Design guide RNAs targeting the C-terminus of endogenous C16orf70

• Include a repair template containing fluorescent protein tag sequence

• Perform CRISPR/Cas9-mediated knock-in

• Validate tagged protein expression by western blot using C16orf70 antibodies

• Conduct live-cell imaging to track C16orf70 during autophagosome formation

• Co-express fluorescently tagged BCAS3 to monitor interaction dynamics

Immunoprecipitation-Mass Spectrometry (IP-MS):

• Perform IP using validated C16orf70 antibodies (such as rabbit recombinant monoclonal antibodies)

• Process samples for mass spectrometry analysis

• Identify interaction partners beyond BCAS3

• Validate key interactions with reciprocal IP experiments

• Map interaction domains using truncated protein constructs

These approaches provide complementary data on spatial, temporal, and molecular aspects of C16orf70-BCAS3 interactions during autophagosome formation, offering insights into the regulatory mechanisms of autophagy.

What are the optimal sample preparation techniques for C16orf70 antibody-based immunohistochemistry?

Effective sample preparation is crucial for successful C16orf70 immunohistochemistry. The following protocol has been optimized based on published methodologies:

Tissue preparation and fixation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard histological procedures

• Section tissues at 4-5 μm thickness

• Mount on positively charged slides

• Dry sections overnight at 37°C

Antigen retrieval optimization:

• Deparaffinize sections in xylene (2 × 10 minutes)

• Rehydrate through graded alcohols to water

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95-98°C for 20 minutes

  • Note: This method has shown superior results for C16orf70 compared to EDTA buffer (pH 9.0)

• Allow slides to cool in buffer for 20 minutes

• Wash in PBS (3 × 5 minutes)

Staining protocol:

• Block endogenous peroxidase with 3% H₂O₂ (10 minutes)

• Perform protein blocking with 5% normal goat serum (30 minutes)

• Incubate with primary C16orf70 antibody at the recommended dilution (1:200-1:500) overnight at 4°C

• Wash in PBS (3 × 5 minutes)

• Apply HRP-conjugated secondary antibody (30 minutes at room temperature)

• Develop with DAB chromogen (3-5 minutes)

• Counterstain with hematoxylin

• Dehydrate, clear, and mount

This protocol has been validated for optimal C16orf70 detection across multiple tissue types, with particularly strong signals observed in brain tissues, consistent with the protein's known expression patterns.

How can researchers optimize C16orf70 antibody conditions for multi-color immunofluorescence with autophagy markers?

Multi-color immunofluorescence combining C16orf70 with autophagy markers requires careful optimization of antibody panels, incubation conditions, and imaging parameters:

Antibody panel optimization:

Sequential staining protocol:

• Fix cells with 4% paraformaldehyde (15 minutes, room temperature)

• Permeabilize with 0.1% Triton X-100 (10 minutes)

• Block with 5% BSA containing 10% normal donkey serum (1 hour)

• Incubate with primary C16orf70 antibody at 0.25-2 μg/mL (overnight, 4°C)

• Wash with PBS (3 × 5 minutes)

• Add second primary antibody (LC3B) (4 hours, room temperature)

• Wash with PBS (3 × 5 minutes)

• Add third primary antibody (BCAS3) (4 hours, room temperature)

• Wash with PBS (3 × 5 minutes)

• Incubate with all secondary antibodies simultaneously (1 hour, room temperature, protected from light)

• Wash with PBS (3 × 10 minutes)

• Counterstain nuclei with DAPI (5 minutes)

• Mount with anti-fade mounting medium

Signal amplification for low-abundance targets:
For enhanced detection of C16orf70 in cells with low expression, incorporate a tyramide signal amplification (TSA) step after the primary antibody incubation, which can increase signal intensity by 10-100 fold without increasing background.

Imaging considerations:
Use spectral unmixing during confocal microscopy to eliminate channel bleed-through, particularly important when examining co-localization of C16orf70 with autophagy markers.

What are the key considerations for using C16orf70 antibodies in primary neuronal cultures versus established cell lines?

Working with C16orf70 antibodies in primary neuronal cultures presents unique challenges compared to established cell lines:

Protocol modifications for primary neurons:

• Fixation adjustments:

  • Use 4% paraformaldehyde with 4% sucrose to preserve neuronal morphology

  • Reduce fixation time to 10 minutes to prevent overfixation

  • Perform at room temperature to maintain membrane integrity

• Permeabilization optimization:

  • Use 0.1% Triton X-100 for 5 minutes (reduced from standard 10 minutes)

  • Alternative: 0.05% saponin for more gentle permeabilization

• Blocking enhancements:

  • Increase blocking time to 2 hours

  • Use 10% normal goat serum with 0.1% BSA and 0.3% Triton X-100

  • Add 0.1% glycine to reduce autofluorescence

• Antibody dilutions and incubations:

  • For C16orf70 antibodies: Use at the upper end of recommended range (2 μg/mL for IF)

  • Extend primary antibody incubation to 48 hours at 4°C

  • Include 0.1% Triton X-100 and 5% normal goat serum in antibody dilution buffer

Critical differences from cell lines:

Validation controls specific for neuronal cultures:

• Include C16orf70 knockout/knockdown neurons as negative controls

• Perform pre-absorption with immunizing peptide

• Compare staining pattern with published neuronal expression data

• Use multiple antibodies targeting different epitopes of C16orf70

These protocol adaptations enhance detection sensitivity and specificity in primary neurons, where C16orf70's role in autophagy may have specialized functions compared to established cell lines.

How should researchers design experiments to investigate C16orf70's role in selective versus non-selective autophagy?

To differentiate C16orf70's function in selective versus non-selective autophagy, a systematic experimental design approach is required:

Experimental framework:

• Baseline characterization:

  • Immunofluorescence co-localization of C16orf70 with LC3B (general autophagy marker)

  • Western blot analysis of C16orf70 expression under basal conditions

  • Co-immunoprecipitation with BCAS3 to confirm complex formation

• Non-selective autophagy induction:

  • Nutrient starvation (EBSS medium, 2-6 hours)

  • mTOR inhibition (rapamycin, 100 nM, 6-24 hours)

  • Track C16orf70 localization using validated antibodies at 0.25-2 μg/mL concentration

  • Quantify co-localization with phagophore markers

• Selective autophagy pathways:

  Mitophagy:

  • Induce with CCCP (10 μM, 12 hours) or Oligomycin/Antimycin A combination

  • Analyze C16orf70 co-localization with PINK1, Parkin, and TOM20

  Aggrephagy:

  • Induce with proteasome inhibitors (MG132, 5 μM, 16 hours)

  • Examine C16orf70 association with p62/SQSTM1 and ubiquitin-positive aggregates

  Xenophagy:

  • Infect cells with GFP-tagged bacteria

  • Assess C16orf70 recruitment to bacteria-containing autophagosomes

• Functional analysis:

  • CRISPR/Cas9 knockout of C16orf70

  • siRNA knockdown (with validation using C16orf70 antibodies)

  • Track autophagy flux using tandem mRFP-GFP-LC3 reporters

  • Measure selective substrate degradation (e.g., mtDNA for mitophagy, aggregated proteins for aggrephagy)

This comprehensive approach allows researchers to distinguish pathway-specific roles of C16orf70, potentially revealing specialized functions in different types of autophagy.

What methods can be used to quantitatively assess changes in C16orf70 expression and localization during cellular stress responses?

Quantitative assessment of C16orf70 during stress responses requires multi-modal analytical approaches:

Western blot quantification:

• Expose cells to stressors (oxidative stress: H₂O₂, 200 μM; ER stress: tunicamycin, 2 μg/mL; hypoxia: 1% O₂)

• Prepare cell lysates at multiple time points (0, 2, 4, 8, 12, 24 hours)

• Perform western blot using C16orf70 antibody at 0.04-0.4 μg/mL

• Normalize to housekeeping proteins (GAPDH, β-actin)

• Quantify band intensity using ImageJ or similar software

• Calculate fold change relative to untreated controls

High-content imaging analysis:

• Seed cells in 96-well imaging plates

• Apply stress conditions in technical triplicates

• Fix and immunostain with C16orf70 antibody (0.25-2 μg/mL)

• Co-stain with markers for:

  • Autophagosome formation (LC3B)

  • Stress granules (G3BP1)

  • Organelle markers (mitochondria, ER, Golgi)

• Perform automated image acquisition (≥9 fields/well)

• Analyze using CellProfiler or similar software for:

  • Mean C16orf70 intensity

  • Subcellular distribution (nuclear/cytoplasmic ratio)

  • Co-localization coefficients (Pearson's, Mander's)

  • Puncta formation (size, number, intensity)

Flow cytometry for population analysis:

• Fix and permeabilize cells after stress treatment

• Stain with fluorophore-conjugated C16orf70 antibody

• Perform flow cytometry analysis

• Gate populations based on C16orf70 expression levels

• Correlate with cell cycle phases or viability markers

qRT-PCR for transcriptional regulation:

• Extract RNA from stressed cells

• Perform reverse transcription

• Quantify C16orf70 mRNA using specific primers

• Normalize to reference genes

• Correlate transcriptional and protein-level changes

This multi-parameter analysis provides comprehensive insights into stress-induced changes in C16orf70 expression, localization, and potential post-translational modifications.

How should researchers interpret conflicting C16orf70 antibody results across different experimental platforms?

When faced with conflicting results using C16orf70 antibodies across different platforms, researchers should implement a systematic troubleshooting and reconciliation approach:

Antibody-specific factors:

• Epitope mapping analysis:

  • Compare immunogen sequences of different antibodies

  • For example, HPA041214 targets "MLDLEVVPERSLGNEQWEFTLGMPLAQAVAIL..." while HPA041131 targets "HGATVKRMYIYSGNSLQDTKAPMMPLSCFLGNVY..."

  • Different epitopes may be differentially accessible in various applications

• Antibody format considerations:

  • Polyclonal antibodies (like HPA041214 and HPA041131) recognize multiple epitopes

  • Monoclonal antibodies (like EPR14035) target a single epitope

  • Reconcile results by using antibodies detecting distinct regions

Technical platform reconciliation:

Biological vs. technical variation assessment:

• Run side-by-side comparisons using the same samples

• Include appropriate positive controls (recombinant C16orf70 protein)

• Validate with orthogonal methods (mass spectrometry, RNA sequencing)

• Consider cell/tissue-specific post-translational modifications

• Assess splice variant detection specificity

Recommendation for resolution:
When conflicting results persist, implement a validation hierarchy:

• Confirm specificity using knockout/knockdown controls

• Prioritize results from multiple antibodies targeting different epitopes

• Weight evidence based on technical quality and reproducibility

• Consider biological context (cell type, experimental conditions)

• Report discrepancies transparently in publications

This systematic approach helps distinguish genuine biological variation from technical artifacts when interpreting conflicting C16orf70 antibody results.

What criteria should be used to evaluate C16orf70 antibody performance in specialized applications?

Evaluating C16orf70 antibody performance for specialized applications requires application-specific criteria and validation metrics:

For super-resolution microscopy:

• Signal-to-noise ratio assessment:

  • Calculate quantitative SNR values in test samples

  • Minimum acceptable SNR: >5:1 for STED, SIM; >10:1 for STORM/PALM

  • C16orf70 antibodies should be tested at multiple concentrations (0.25-4 μg/mL)

• Localization precision:

  • Measure point spread function using sub-diffraction beads

  • Calculate mean localization precision

  • Expected values: <30 nm for STORM/PALM applications

• Photostability evaluation:

  • Assess fluorophore bleaching rates

  • Determine optimal imaging parameters

  • Consider direct immunofluorescence to reduce distance to target

For proximity ligation assays (PLA):

• Antibody compatibility testing:

  • Confirm species compatibility of C16orf70 antibody pairs

  • Verify performance in standard immunofluorescence first

  • Optimize C16orf70 antibody concentration range (0.25-2 μg/mL)

• Controls required:

  • Positive control: Known C16orf70 interaction partner (BCAS3)

  • Negative controls: Single antibody controls, non-expressed protein pairs

  • Biological relevance validation: siRNA knockdown reduces signal

• Signal quantification metrics:

  • PLA dots per cell (mean ± SD)

  • Nuclear vs. cytoplasmic distribution

  • Co-localization with autophagosome markers

For FACS/cell sorting applications:

• Titration optimization:

  • Serial dilutions to determine optimal concentration

  • Evaluation of separation index between positive/negative populations

  • Detection of endogenous vs. overexpressed C16orf70

• Fluorophore selection criteria:

  • Brightness and photostability

  • Compatibility with other panel markers

  • Minimal spectral overlap

• Validation requirements:

  • Comparison with isotype controls

  • Blocking with immunizing peptide

  • Correlation with alternative detection methods

These specialized criteria ensure that C16orf70 antibodies are appropriately validated for advanced applications, improving data reliability and interpretation.

What are the current limitations and future directions for C16orf70 antibody-based research?

Current limitations in C16orf70 antibody research present opportunities for methodological advancement and expanded applications:

Current technical limitations:

• Epitope accessibility issues:

  • Many available antibodies target similar regions of C16orf70

  • Limited antibodies against conformational epitopes

  • Potential masking of epitopes during protein-protein interactions

• Specificity across species:

  • Most validated C16orf70 antibodies are optimized for human samples

  • Cross-reactivity with murine or other model organisms is poorly characterized

  • Verification in non-human models requires additional validation steps

• Post-translational modification detection:

  • Limited availability of modification-specific antibodies

  • Phosphorylation, ubiquitination states difficult to discriminate

  • Functional significance of modifications remains understudied

Emerging research directions:

• Development of proximity-based labeling approaches:

  • Integration of C16orf70 antibodies with BioID or APEX2 technologies

  • Mapping dynamic interactomes during autophagy progression

  • Temporal resolution of protein complex assembly

• Single-cell analysis applications:

  • Adaptation of C16orf70 antibodies for CyTOF/mass cytometry

  • Integration with single-cell RNA sequencing data

  • Heterogeneity assessment in autophagy responses

• Therapeutic targeting potential:

  • C16orf70 as a biomarker for autophagy dysregulation

  • Development of conformation-specific antibodies

  • Combination with small molecule modulators of autophagy

Methodological innovations needed:

• Increased antibody diversity:

  • Development of antibodies against diverse epitopes

  • Humanized antibodies for therapeutic applications

  • Non-immunoglobulin binding scaffolds (nanobodies, affimers)

• Spatiotemporal dynamics:

  • Photoactivatable antibody fragments

  • Intracellular delivery systems for live-cell imaging

  • Correlative light-electron microscopy compatible detection

These advancements will address current limitations and expand the utility of C16orf70 antibodies in both basic research and potential clinical applications, particularly in disorders involving autophagy dysregulation.

How does understanding C16orf70's role in autophagy contribute to broader research on neurodegenerative diseases?

C16orf70's function in autophagy regulation has significant implications for neurodegenerative disease research, with antibody-based studies providing critical mechanistic insights:

Pathological connections:

• Protein aggregation disorders:

  • C16orf70's role in selective autophagy suggests involvement in clearing protein aggregates

  • Antibody-based co-localization studies show association with aggregated proteins

  • Potential role in Alzheimer's, Parkinson's, and Huntington's disease pathophysiology

• Autophagy dysfunction in neurodegeneration:

  • C16orf70-BCAS3 complex formation is critical for autophagosome biogenesis

  • Dysregulation may contribute to accumulation of dysfunctional mitochondria

  • Impaired autophagic flux linked to neuronal cell death

• Stress response integration:

  • C16orf70 responds to cellular stressors common in neurodegenerative conditions

  • May function as a stress sensor in the autophagy pathway

  • Potential therapeutic target for enhancing neuronal resilience

Research applications in neurodegeneration models:

• Patient-derived samples:

  • Immunohistochemical analysis of C16orf70 in post-mortem brain tissue

  • Comparison of expression/localization between healthy and disease states

  • Correlation with disease severity and progression markers

• Animal models:

  • C16orf70 knockout/knockdown effects on neurodegeneration progression

  • Antibody-based tracking of autophagy dynamics in vivo

  • Therapeutic modulation of C16orf70-mediated pathways

• iPSC-derived neurons:

  • Disease modeling using patient-specific neurons

  • C16orf70 antibody-based high-content screening

  • Identification of small molecules that modulate C16orf70 function

Translational potential:

Antibody-based research on C16orf70 contributes to potential therapeutic strategies:

• Gene therapy approaches targeting C16orf70 expression

• Small molecule modulators of C16orf70-BCAS3 interaction

• Biomarker development for autophagy dysfunction

• Combinatorial approaches targeting multiple autophagy regulators