atp6v1c1a Antibody

What is ATP6V1C1A and what cellular functions does it participate in?

ATP6V1C1A is the zebrafish ortholog of the human ATP6V1C1 gene, which encodes a component of vacuolar ATPase (V-ATPase), a multisubunit enzyme that mediates acidification of intracellular compartments . The protein serves as part of the V1 complex responsible for ATP hydrolysis, working in conjunction with the membrane-integrated V0 complex that translocates protons .

In cellular systems, ATP6V1C1A participates in:

• Lysosomal acidification and pH maintenance

• Vesicular trafficking

• Autophagy regulation

• Ion homeostasis in specialized cells

Research has demonstrated that alterations in ATP6V1C1 can lead to disrupted lysosomal morphology and function, resulting in defective autophagic flux and accumulation of lysosomal substrates . This suggests the zebrafish ortholog likely plays similar critical roles in vesicular systems.

What are the primary applications for ATP6V1C1A antibodies in zebrafish research?

ATP6V1C1A antibodies serve multiple critical applications in zebrafish research, each requiring specific methodological considerations:

When designing experiments with ATP6V1C1A antibodies in zebrafish, it's essential to validate reactivity specifically with Danio rerio samples, as most commercial antibodies were initially developed against human or mouse proteins . Cross-species validation is critical before proceeding with extensive experimental series.

How should researchers select the appropriate ATP6V1C1A antibody for their experimental design?

Selection of an appropriate ATP6V1C1A antibody requires consideration of multiple technical factors:

• Epitope specificity: Choose antibodies targeting conserved regions between human ATP6V1C1 and zebrafish ATP6V1C1A. The N-terminal region (amino acids 1-110) is often suitable for cross-species applications .

• Application compatibility: Verify the antibody has been validated for your specific application. Some antibodies perform well in Western blotting but poorly in immunohistochemistry applications .

• Clonality considerations:

  • Polyclonal antibodies offer broader epitope recognition but potential batch variation

  • Monoclonal antibodies provide consistency but may have limited epitope accessibility in certain applications

• Host species compatibility: Select antibody host species that won't conflict with other antibodies in multi-labeling experiments. Rabbit-derived polyclonal antibodies are commonly available for ATP6V1C1A detection .

When examining commercially available options, review published literature using these antibodies in zebrafish models to assess performance consistency across laboratories.

How can ATP6V1C1A antibodies be utilized to investigate lysosomal disorders in zebrafish models?

Investigating lysosomal disorders in zebrafish using ATP6V1C1A antibodies requires sophisticated experimental approaches:

• Subcellular co-localization protocol:

  • Seed primary zebrafish cells at 20×10³ density on 12-μm cover glasses

  • After 24 hours in complete medium, fix with 3% PFA (30 minutes at 4°C)

  • Permeabilize with 0.5% Triton X-100 (10 minutes at room temperature)

  • Co-stain with anti-ATP6V1C1A antibody and lysosomal marker antibodies (e.g., anti-LAMP1)

  • Analyze using confocal microscopy to assess morphology and localization patterns

• Functional assays to assess lysosomal acidification:

  • LysoTracker staining to visualize acidic organelles

  • LysoSensor probes to measure intra-lysosomal pH changes

  • Correlation of ATP6V1C1A immunostaining with acidification patterns

Research has demonstrated that pathogenic variants in ATP6V1C1 result in gain-of-function mechanisms that upregulate V-ATPase activity, leading to increased lysosomal acidification . Zebrafish models provide an excellent system to investigate how these alterations affect developmental processes.

What methodological approaches are recommended for investigating the role of ATP6V1C1A in neurodevelopmental disorders?

Recent research has identified ATP6V1C1 variants associated with neurodevelopmental phenotypes resembling DOORS syndrome . To investigate similar roles for ATP6V1C1A in zebrafish neurodevelopment:

• Morpholino knockdown approach:

  • Design morpholinos targeting ATP6V1C1A splice sites or translation start sites

  • Inject 1-2 nl of morpholino (0.25-1.0 mM) into 1-4 cell stage embryos

  • Validate knockdown efficacy using ATP6V1C1A antibodies via Western blotting

  • Analyze neurodevelopmental phenotypes through behavioral assays and morphological assessment

• CRISPR-Cas9 gene editing protocol:

  • Design guide RNAs targeting conserved regions of ATP6V1C1A

  • Co-inject with Cas9 protein into single-cell embryos

  • Screen F0 embryos for mutations and phenotypes

  • Establish stable mutant lines for comprehensive phenotypic characterization

  • Validate protein loss using ATP6V1C1A antibodies

• Rescue experiments:

  • Co-inject human ATP6V1C1 mRNA with morpholinos or after CRISPR editing

  • Evaluate rescue efficacy through phenotypic assessment and ATP6V1C1A antibody staining

  • Test variant forms associated with human disorders to assess functional conservation

This comprehensive approach allows for detailed investigation of how ATP6V1C1A disruption affects neuronal development, providing insights into conserved mechanisms between zebrafish and human neurodevelopmental disorders.

What controls are essential when conducting immunoprecipitation with ATP6V1C1A antibodies?

Immunoprecipitation studies with ATP6V1C1A antibodies require rigorous controls:

• Input controls:

  • Reserve 5-10% of pre-IP lysate to verify target protein presence

  • Use in parallel with IP samples for Western blot analysis

• Negative controls:

  • Isotype-matched non-specific IgG from the same host species as the ATP6V1C1A antibody

  • This controls for non-specific binding to the antibody or beads

  • Process identically to experimental samples

• Specificity controls:

  • Pre-incubation of antibody with recombinant ATP6V1C1A protein

  • Should significantly reduce or eliminate target band in Western blots

  • Validates specificity of the IP procedure

• Procedural protocol:

  • Lyse 1mg of zebrafish tissue in non-denaturing buffer

  • Pre-clear with Protein A/G beads (1 hour at 4°C)

  • Incubate cleared lysate with ATP6V1C1A antibody (1:50 dilution) overnight at 4°C

  • Add fresh beads and incubate 4 hours at 4°C

  • Wash 4-5 times with cold IP buffer

  • Elute and analyze by Western blotting using 1:1000 dilution of ATP6V1C1A antibody

This approach has been validated for related V-ATPase subunits and can be adapted for ATP6V1C1A in zebrafish samples with appropriate optimization.

How should researchers troubleshoot cross-reactivity issues with ATP6V1C1A antibodies?

Cross-reactivity presents a significant challenge when working with ATP6V1C1A antibodies, particularly in zebrafish models. Implement this systematic troubleshooting approach:

• Epitope analysis:

  • Perform sequence alignment between zebrafish ATP6V1C1A and potential cross-reactive proteins

  • Identify regions of high homology that might contribute to non-specific binding

  • Consider antibodies raised against unique protein regions

• Blocking optimization:

  • Test gradient concentrations of blocking agents (5-10% non-fat dry milk or BSA)

  • Include 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Consider adding 5% serum from the secondary antibody host species

• Validation with genetic models:

  • Use zebrafish ATP6V1C1A knockout or knockdown lines as negative controls

  • Overexpression models as positive controls

  • These genetic validations provide definitive evidence of antibody specificity

• Peptide competition assays:

  • Pre-incubate antibody with 5-10 fold molar excess of immunizing peptide

  • Compare staining patterns with and without peptide competition

  • True ATP6V1C1A signal should be specifically competed away

When persistent cross-reactivity occurs, consider using epitope-tagged constructs in transgenic models as an alternative approach to directly detect the protein of interest.

What are the optimal protocols for analyzing ATP6V1C1A expression in different zebrafish developmental stages?

Developmental analysis of ATP6V1C1A requires stage-specific protocols:

• Embryonic stages protocol (0-72 hpf):

  • Collect 30-50 embryos per timepoint

  • Dechorionate manually or with pronase treatment

  • Homogenize in RIPA buffer with protease inhibitors

  • For Western blotting, load 20-30 μg protein per lane

  • Use 1:1000-1:2000 dilution of ATP6V1C1A antibody

  • GAPDH serves as an appropriate loading control

• Larval stages protocol (3-30 dpf):

  • Process 10-20 larvae per timepoint

  • For immunohistochemistry, fix in 4% PFA overnight at 4°C

  • Embedding and sectioning: 5-8 μm thickness optimal

  • Antigen retrieval: citrate buffer pH 6.0 or TE buffer pH 9.0

  • Antibody dilution: 1:20-1:200 range, optimize empirically

• Adult tissue protocol:

  • Dissect specific tissues of interest (brain, kidney, etc.)

  • Flash-freeze in liquid nitrogen for protein extraction

  • For immunofluorescence of tissue sections, process as for larvae

  • Counter-staining with DAPI facilitates tissue architecture visualization

• Data analysis considerations:

  • Normalize expression to reliable housekeeping genes/proteins

  • Analyze minimum 3 biological replicates per developmental stage

  • Apply appropriate statistical tests for temporal expression patterns

This comprehensive approach enables detailed characterization of ATP6V1C1A expression dynamics throughout zebrafish development.

How can ATP6V1C1A antibodies be utilized in studying V-ATPase complex assembly and stoichiometry?

Investigating V-ATPase complex assembly requires sophisticated biochemical approaches:

• Blue Native PAGE protocol:

  • Extract membrane fractions from zebrafish tissues in non-denaturing conditions

  • Solubilize with 1% digitonin or 1% n-dodecyl β-D-maltoside

  • Separate on 3-12% gradient Native PAGE gels

  • Transfer to PVDF membranes

  • Probe with ATP6V1C1A antibody (1:500) to detect intact complexes

  • Compare migration patterns with known complex sizes

• Co-immunoprecipitation strategy:

  • Use ATP6V1C1A antibody (1:50 dilution) for pull-down

  • Analyze co-precipitated proteins by mass spectrometry or Western blotting

  • Probe for other V-ATPase components (ATP6V1A, ATP6V1B2)

  • Different detergent conditions can reveal subcomplex interactions

• Proximity labeling approach:

  • Generate ATP6V1C1A-BioID or ATP6V1C1A-APEX2 fusion constructs

  • Express in zebrafish cells or transgenic models

  • Activate labeling and purify biotinylated proteins

  • Identify interaction partners through mass spectrometry

  • Validate with ATP6V1C1A antibody immunoblotting

• Quantitative stoichiometry analysis:

  • Use purified recombinant ATP6V1C1A protein standards

  • Generate calibration curves for absolute quantification

  • Compare stoichiometric ratios across different tissues and conditions

  • Analyze how pathogenic variants affect complex assembly

These methodologies provide comprehensive insights into how ATP6V1C1A integrates into the V-ATPase complex and how this assembly may be altered in disease states.

How should researchers interpret discrepancies between ATP6V1C1A antibody results and gene expression data?

Discrepancies between protein detection and gene expression require systematic analysis:

• Validation of antibody specificity:

  • Perform Western blotting on samples with confirmed ATP6V1C1A expression

  • Include positive controls (human or mouse samples with cross-reactive antibodies)

  • Test multiple antibodies targeting different epitopes when available

• Post-transcriptional regulation assessment:

  • Measure mRNA stability through actinomycin D chase experiments

  • Analyze microRNA targeting ATP6V1C1A through bioinformatic prediction and validation

  • Evaluate protein half-life through cycloheximide chase assays

• Technical considerations:

  • Compare protein extraction methods (RIPA vs. urea-based buffers)

  • Test different fixation protocols for immunohistochemistry

  • Optimize antigen retrieval methods (citrate vs. TE buffer)

• Biological interpretation framework:

  • Consider tissue-specific post-transcriptional regulation

  • Evaluate developmental stage-specific regulation mechanisms

  • Assess potential protein localization changes affecting detection

When persistent discrepancies exist, consider using orthogonal methods such as mass spectrometry-based proteomics to provide antibody-independent validation of protein expression patterns.

What methodological approaches are recommended for quantifying changes in ATP6V1C1A expression under experimental conditions?

Accurate quantification of ATP6V1C1A expression changes requires rigorous methodological approaches:

• Western blot quantification protocol:

  • Use gradient protein loadings to establish linear detection range

  • Include recombinant protein standards at known concentrations

  • Apply fluorescent secondary antibodies for wider linear range

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

  • Use LiCor Odyssey or similar quantitative imaging systems

• Immunofluorescence quantification:

  • Acquire images with identical exposure settings across all samples

  • Use automated image analysis with consistent thresholding

  • Measure total integrated intensity per cell or structure

  • Analyze minimum 50-100 cells per condition across 3+ biological replicates

• Flow cytometry approach:

  • Permeabilize fixed cells with 0.1% saponin

  • Stain with ATP6V1C1A antibody (optimal dilution determined empirically)

  • Use fluorescent secondary antibodies

  • Include isotype controls and secondary-only controls

  • Measure median fluorescence intensity for population analysis

• Recommended statistical analysis:

  • Apply appropriate normality tests before choosing statistical methods

  • Use ANOVA with post-hoc tests for multiple condition comparisons

  • Report effect sizes alongside p-values

  • Include power analysis to justify sample sizes

This comprehensive approach ensures robust quantification of ATP6V1C1A expression changes under various experimental conditions.

How can researchers integrate ATP6V1C1A antibody data with functional assays to investigate V-ATPase activity?

Integrating ATP6V1C1A localization and expression data with functional V-ATPase assays provides mechanistic insights:

• Lysosomal pH measurement protocol:

  • Load cells with pH-sensitive dyes (LysoSensor Yellow/Blue DND-160)

  • Calibrate with ionophores in buffers of known pH

  • Correlate pH measurements with ATP6V1C1A immunostaining patterns

  • Analyze how experimental manipulations affect both protein expression and function

• V-ATPase activity assay:

  • Isolate membrane fractions from zebrafish tissues

  • Measure ATP hydrolysis through phosphate release assays

  • Determine bafilomycin A1-sensitive component (specific to V-ATPase)

  • Correlate activity with ATP6V1C1A protein levels determined by immunoblotting

• Genetic manipulation validation:

  • Knockdown ATP6V1C1A using morpholinos or CRISPR-Cas9

  • Confirm protein reduction via ATP6V1C1A antibody staining

  • Measure functional consequences on organelle acidification

  • Perform rescue experiments with wild-type and mutant constructs

• Integrative data analysis approach:

  • Plot correlations between ATP6V1C1A levels and functional readouts

  • Apply multivariate analysis to identify key variables affecting V-ATPase function

  • Develop mathematical models integrating protein expression and activity data

  • Use Bayesian statistical frameworks to incorporate prior knowledge from literature

This integrative approach provides a comprehensive understanding of how ATP6V1C1A expression and localization relate to functional V-ATPase activity in physiological and pathological contexts.