ATP6V1D Antibody, HRP conjugated

What is ATP6V1D and why is it an important research target?

ATP6V1D functions as a subunit of the peripheral V1 complex of vacuolar ATPase (V-ATPase), which is responsible for acidifying various intracellular compartments in eukaryotic cells. This acidification provides essential energy for transport processes within the vacuolar system . Beyond its conventional role, ATP6V1D has recently been identified as a key metabolic regulator of hepatocellular carcinoma (HCC) stemness, with elevated expression correlating with poor clinical outcomes in HCC patients . This dual role in normal cellular physiology and disease pathogenesis makes ATP6V1D a critical target for both basic science investigations and translational research exploring potential therapeutic interventions.

What is the functional significance of using HRP-conjugated ATP6V1D antibodies versus other conjugates?

HRP (Horseradish Peroxidase) conjugation provides significant advantages for detection sensitivity and versatility in multiple applications. When using HRP-conjugated ATP6V1D antibodies, researchers can employ various substrates that produce colorimetric, chemiluminescent, or fluorescent signals, offering flexibility based on available instrumentation and experimental needs . The enzyme amplification step enables detection of low abundance ATP6V1D protein in complex biological samples. Unlike FITC-conjugated antibodies which provide direct fluorescence , HRP conjugates require a substrate development step but typically offer greater sensitivity and signal stability. This is particularly valuable when investigating ATP6V1D expression in clinical samples where protein quantities may be limited or when examining subtle changes in expression levels during experimental manipulations.

How should researchers design experiments to validate ATP6V1D antibody specificity?

Validating antibody specificity is critical for ensuring reliable experimental results. A comprehensive validation protocol should include:

• Positive and negative control tissues/cells: Use samples with known high expression (HCC cell lines) and low/no expression of ATP6V1D .

• Knockdown/knockout validation: Compare staining between wildtype and ATP6V1D knockdown samples generated using siRNA or CRISPR-Cas9 approaches .

• Peptide competition assay: Pre-incubate the antibody with recombinant ATP6V1D protein (1-247AA) before application to verify signal reduction.

• Western blot analysis: Confirm single band at expected molecular weight (~34kDa) before proceeding to other applications.

• Cross-reactivity assessment: Test the antibody on samples from different species to confirm human specificity .

For HRP-conjugated antibodies specifically, include substrate-only controls to differentiate between specific signal and background peroxidase activity in tissues.

What are the optimal experimental conditions for using ATP6V1D Antibody, HRP conjugated in ELISA assays?

Optimizing ELISA protocols for ATP6V1D detection requires careful consideration of several parameters:

Recommended ELISA Protocol:

• Coating: Use 1-5 μg/ml of capture antibody in carbonate buffer (pH 9.6), incubate overnight at 4°C.

• Blocking: 3% BSA in PBS for 1-2 hours at room temperature.

• Sample preparation: Cell/tissue lysates should be prepared in a buffer containing 0.01M PBS, pH 7.4 with protease inhibitors .

• Antibody dilution: Start with 1:1000 dilution of the HRP-conjugated ATP6V1D antibody and optimize as needed.

• Incubation conditions: 1-2 hours at room temperature or overnight at 4°C in 1% BSA in PBS-T (PBS + 0.05% Tween-20).

• Substrate: TMB substrate for 15-30 minutes, followed by stop solution.

• Storage handling: Maintain antibody at -20°C or -80°C and avoid repeated freeze-thaw cycles .

Sensitivity can be further enhanced by extending incubation times or using signal amplification systems. Always include standard curves using recombinant ATP6V1D protein (1-247AA) to ensure quantitative accuracy and reproducibility.

How can ATP6V1D Antibody, HRP conjugated be utilized to investigate autophagy regulation in cancer research?

Recent research has established ATP6V1D as a crucial regulator of autophagic flux in hepatocellular carcinoma . To investigate this connection:

• Dual immunostaining approach: Combine ATP6V1D antibody with autophagy markers (LC3, p62) to visualize colocalization. For HRP-conjugated antibodies, use sequential detection with different substrates that yield distinguishable colors.

• Autophagy flux assessment: Measure ATP6V1D levels by western blot or ELISA under conditions of autophagy stimulation (starvation, rapamycin) or inhibition (3-MA, bafilomycin A₁) .

• Lysosomal acidification analysis: Use ATP6V1D antibody to correlate protein expression with lysosomal pH measured by LysoTracker or acridine orange. The HRP-conjugated antibody can be used in western blot or ELISA to quantify expression levels.

• Interaction studies: Investigate ATP6V1D's role in autophagosome-lysosome fusion by examining its interaction with CHMP4B and IST1 to foster ESCRT-III complex assembly .

• Therapeutic intervention studies: Monitor changes in ATP6V1D expression following treatment with low-dose bafilomycin A₁ or other V-ATPase inhibitors to correlate with therapeutic efficacy in cancer models .

This multifaceted approach enables researchers to comprehensively characterize ATP6V1D's mechanistic role in maintaining autophagic flux and its contribution to cancer stemness and progression.

What methods can be employed to investigate the relationship between ATP6V1D expression and clinical outcomes in cancer patients?

To establish clinically relevant correlations between ATP6V1D expression and patient outcomes:

When using HRP-conjugated antibodies, standardize detection methods across all samples to ensure comparable staining intensities for accurate quantification and statistical analysis.

How can researchers distinguish between functionally active and inactive ATP6V1D in experimental systems?

Distinguishing functionally active ATP6V1D from inactive forms requires sophisticated approaches beyond mere protein detection:

• V-ATPase activity assays: Measure proton pumping activity in isolated vesicles or lysosomes and correlate with ATP6V1D levels detected by the HRP-conjugated antibody.

• Conformational state analysis: Use limited proteolysis followed by detection with domain-specific antibodies to determine structural states associated with activation.

• ATP binding and hydrolysis: Develop coupled enzyme assays to measure ATPase activity in samples immunoprecipitated with ATP6V1D antibodies.

• Protein-protein interaction mapping: Investigate ATP6V1D's association with other V-ATPase subunits or regulatory proteins using proximity ligation assays or co-immunoprecipitation followed by detection with HRP-conjugated antibodies .

• Post-translational modification detection: Combine ATP6V1D immunoprecipitation with phosphorylation-specific antibodies to identify activation-associated modifications.

These approaches can help researchers determine not just the presence of ATP6V1D but its functional status within the V-ATPase complex, providing deeper insights into its regulatory mechanisms.

What are the critical factors affecting reproducibility when using ATP6V1D Antibody, HRP conjugated in different experimental platforms?

Ensuring reproducibility across different experimental platforms requires attention to several critical factors:

Additionally, researchers should develop standard operating procedures (SOPs) that detail exact conditions for each experimental platform and maintain detailed records of antibody source, lot number, and validation results to maximize reproducibility.

How can researchers optimize the detection of low-abundance ATP6V1D in clinical samples?

Detecting low-abundance ATP6V1D in clinical samples presents significant challenges that can be addressed through several optimization strategies:

• Signal amplification systems: Employ tyramide signal amplification (TSA) techniques compatible with HRP-conjugated antibodies to enhance detection sensitivity by 10-100 fold.

• Sample enrichment methods: Use immunoprecipitation prior to detection to concentrate ATP6V1D from larger sample volumes.

• Optimized antigen retrieval: For fixed tissues, test multiple antigen retrieval methods to maximize epitope accessibility, including:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0)

  • Protease-based retrieval with optimized enzyme concentration and incubation time

• Extended antibody incubation: Increase antibody incubation time to 24-48 hours at 4°C to enhance binding to low-abundance targets.

• Two-step detection system: For maximum sensitivity, use an unconjugated primary ATP6V1D antibody followed by an HRP-conjugated secondary antibody with polymeric signal amplification.

• Reduced background strategies: Employ specialized blocking reagents to minimize non-specific binding and implement multiple washing steps with optimized salt concentrations.

• Digital image analysis: Utilize computational approaches to enhance signal detection and quantification in immunohistochemistry applications.

These approaches can significantly improve detection of low-abundance ATP6V1D, enabling more accurate assessment of its expression in clinical samples and correlation with disease parameters.

How can ATP6V1D Antibody, HRP conjugated be utilized to investigate the role of ATP6V1D in hepatocellular carcinoma stemness?

Recent research has established ATP6V1D as a key driver of hepatocellular carcinoma stemness . The following methodological approach can be implemented using HRP-conjugated ATP6V1D antibodies:

• Cancer stem cell isolation: Sort liver cancer stem cells (LCSCs) based on established markers (CD133, EpCAM) and compare ATP6V1D expression with non-stem populations using immunoblotting with HRP-conjugated antibodies.

• Sphere formation assays: Correlate ATP6V1D expression levels with tumorsphere formation capacity following knockdown or overexpression experiments.

• RNA-protein correlation: Combine qRT-PCR data on ATP6V1D mRNA with protein detection using HRP-conjugated antibodies to establish transcriptional and post-transcriptional regulation mechanisms.

• In vivo tumor initiation studies: Inject varying numbers of cells with different ATP6V1D expression levels to determine tumor-initiating capacity and correlate with protein expression using the HRP-conjugated antibody on resulting tumors.

• Therapeutic resistance assessment: Monitor ATP6V1D expression changes in response to conventional chemotherapy and correlate with stemness marker expression and resistance development.

This comprehensive approach allows researchers to establish both correlative and causative relationships between ATP6V1D expression and cancer stemness properties, potentially identifying new therapeutic targets.

What experimental approaches can be used to study ATP6V1D's role in lysosomal function and autophagy?

ATP6V1D plays a dual role in promoting lysosomal acidification and facilitating autophagosome-lysosome fusion . To investigate these functions:

• Lysosomal pH measurement: Correlate ATP6V1D expression (detected using HRP-conjugated antibodies) with lysosomal pH using ratiometric fluorescent probes.

• Autophagy flux assessment:

  • Western blot analysis of LC3-II/LC3-I ratio and p62 levels in ATP6V1D knockdown vs. control cells

  • Tandem fluorescent-tagged LC3 (tfLC3) assays to monitor autophagosome-lysosome fusion

• ESCRT-III complex interaction studies:

  • Co-immunoprecipitation experiments to detect ATP6V1D interaction with CHMP4B and IST1

  • Proximity ligation assays to visualize these interactions in situ

• Functional recovery experiments:

  • Rescue experiments using wildtype vs. mutant ATP6V1D in knockout cells

  • Correlation of functional recovery with restoration of autophagy markers

• Pharmacological intervention:

  • Comparison of ATP6V1D knockdown effects with bafilomycin A₁ treatment at varying doses

  • Assessment of combinatorial effects of ATP6V1D modulation with other autophagy regulators

These methods enable detailed characterization of ATP6V1D's mechanistic role in the autophagy-lysosomal pathway, providing insights into both normal cellular physiology and disease pathogenesis.

How can ATP6V1D expression patterns be compared across different tissue types and disease states?

Comparative analysis of ATP6V1D expression across tissues and conditions requires standardized approaches:

• Multi-tissue Western blot analysis: Using HRP-conjugated ATP6V1D antibodies to compare expression levels across tissue lysates, with careful loading control normalization.

• Quantitative immunohistochemistry:

  • Tissue microarray construction containing multiple tissue types and disease states

  • Digital image analysis for objective quantification of staining intensity

  • Statistical comparison across groups with appropriate multiple testing correction

• Database integration approaches:

  • Correlation of experimental data with publicly available transcriptomic and proteomic datasets

  • Integration of clinical metadata to identify disease-specific expression patterns

• Cell type-specific expression analysis:

  • Single-cell techniques to identify cell populations with differential ATP6V1D expression

  • Isolation of specific cell populations, such as V-ATPase B1 subunit (ATP6V1B1) expressing cells from kidney and epididymis

• Standardized reporting:

  • Development of standardized scoring systems for ATP6V1D expression

  • Inclusion of relevant controls and reference standards for cross-study comparisons

This systematic approach allows researchers to generate comprehensive ATP6V1D expression profiles across normal and pathological states, potentially identifying tissue-specific functions and disease associations.

What emerging technologies might enhance the utility of ATP6V1D antibodies in studying V-ATPase biology?

Several cutting-edge technologies offer promising avenues for advancing ATP6V1D research:

• Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy can reveal the precise subcellular localization of ATP6V1D within the V-ATPase complex when using appropriately conjugated antibodies.

• Mass cytometry (CyTOF): Allows simultaneous detection of ATP6V1D alongside dozens of other proteins at single-cell resolution by conjugating antibodies to rare earth metals instead of fluorophores or enzymes.

• Spatial transcriptomics with protein detection: Combining RNA-seq with protein detection using ATP6V1D antibodies can reveal relationships between transcriptional regulation and protein expression in tissue contexts.

• Intrabodies and nanobodies: Development of cell-permeable ATP6V1D-specific antibody fragments that can track and potentially modulate protein function in living cells.

• CRISPR screens with antibody-based readouts: High-throughput functional genomics approaches using ATP6V1D antibodies to detect changes in protein levels or localization following genetic perturbations.

• Cryo-electron microscopy: Structural studies using ATP6V1D antibodies as fiducial markers to determine the protein's position and orientation within the V-ATPase complex.

These technologies can provide unprecedented insights into ATP6V1D biology, potentially revealing new functional roles and therapeutic opportunities in diseases like hepatocellular carcinoma.

How might ATP6V1D research contribute to therapeutic development for diseases involving vacuolar ATPase dysfunction?

ATP6V1D research has significant translational potential across multiple disease areas:

• Targeted cancer therapeutics:

  • Development of specific ATP6V1D inhibitors as alternatives to broad V-ATPase inhibitors like bafilomycin A₁

  • Antibody-drug conjugates targeting ATP6V1D-overexpressing cells

  • Combination therapies targeting ATP6V1D alongside other autophagy regulators

• Biomarker development:

  • Prognostic stratification of HCC patients based on ATP6V1D expression levels

  • Predictive biomarkers for response to therapies targeting the autophagy-lysosomal pathway

  • Monitoring tools for disease progression and treatment response

• Imaging applications:

  • Development of labeled ATP6V1D antibodies for in vivo imaging of tumors with dysregulated V-ATPase activity

  • Correlation of imaging data with clinical outcomes to validate as a non-invasive monitoring tool

• Precision medicine approaches:

  • Patient stratification based on ATP6V1D expression profiles

  • Tailored therapeutic regimens targeting the specific mechanisms of V-ATPase dysfunction

• Autophagy modulation strategies:

  • Fine-tuning of autophagy through selective targeting of ATP6V1D-dependent processes

  • Development of synthetic biology approaches to conditionally regulate ATP6V1D activity

These approaches represent promising avenues for translating basic ATP6V1D research into clinical applications, particularly in oncology and lysosomal storage disorders.

What key technical challenges remain to be solved in ATP6V1D antibody development and application?

Despite significant progress, several technical challenges remain in ATP6V1D research:

• Epitope-specific antibodies: Development of antibodies recognizing specific functional domains or conformational states of ATP6V1D to distinguish between active and inactive forms.

• Cross-species reactivity optimization: Creating antibodies with consistent performance across experimental animal models to facilitate translational research.

• Quantitative standardization: Establishing absolute quantification methods for ATP6V1D to enable meaningful cross-study comparisons.

• Tissue penetration enhancement: Improving antibody formats to achieve better penetration in thick tissue sections or whole-mount preparations.

• Multiplexing capabilities: Developing compatible conjugates and detection systems for simultaneous visualization of ATP6V1D alongside multiple other proteins.

• Live-cell applications: Creating non-toxic, cell-permeable ATP6V1D antibody derivatives for tracking protein dynamics in living systems.

• Automation compatibility: Optimizing antibody performance for high-throughput and automated platforms to facilitate large-scale screening applications.

Addressing these challenges will significantly advance ATP6V1D research capabilities, enabling more sophisticated investigations into its role in normal physiology and disease mechanisms, particularly in cancer stemness and autophagy regulation.