ATP6V1D Antibody, Biotin conjugated

What is ATP6V1D and what is its biological function?

ATP6V1D is a subunit of the V1 complex of vacuolar(H+)-ATPase (V-ATPase), a multisubunit enzyme composed of a peripheral complex (V1) that hydrolyzes ATP and a membrane integral complex (V0) that translocates protons. V-ATPase is responsible for acidifying and maintaining the pH of intracellular compartments. In some cell types, it is targeted to the plasma membrane, where it is responsible for acidifying the extracellular environment. Research indicates ATP6V1D may also play a role in cilium biogenesis through regulation of the transport and localization of proteins to the cilium .

The protein is also known by several aliases including V-type proton ATPase subunit D, V-ATPase subunit D, V-ATPase 28 kDa accessory protein, Vacuolar proton pump subunit D, ATP6M, and VATD. The predicted molecular weight of human ATP6V1D is approximately 28 kDa .

What are the primary research applications for ATP6V1D antibodies?

ATP6V1D antibodies are utilized in multiple research applications depending on their specific conjugation and validation status:

The selection of appropriate application should be based on experimental requirements and the specific validation data available for each antibody conjugate .

What are the optimal storage and handling conditions for biotin-conjugated ATP6V1D antibodies?

Biotin-conjugated ATP6V1D antibodies require specific storage conditions to maintain their activity and specificity:

• Temperature: Store at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles which can compromise antibody integrity

• Short-term storage: For frequent use, store at 4°C for up to one month

• Formulation: Typically supplied in storage buffer containing preservative (e.g., 0.03% Proclin 300) and stabilizers (e.g., 50% Glycerol, PBS, pH 7.4)

When handling the antibody:

• Allow it to equilibrate to room temperature before opening

• Brief centrifugation is recommended before opening the vial

• Working dilutions should be prepared fresh before use and stored at 4°C for short periods only

How does biotin conjugation affect ATP6V1D antibody performance in different applications?

Biotin conjugation offers specific advantages and considerations in research applications:

Advantages:

• Enhanced sensitivity through signal amplification using avidin/streptavidin detection systems

• Increased flexibility in detection methods (can be visualized using various streptavidin-conjugated reporters)

• Potential for multi-layer staining protocols

Application-specific considerations:

• ELISA: Biotin-conjugated antibodies are highly effective due to the strong biotin-streptavidin interaction, providing sensitive detection capabilities

• Flow cytometry: May require careful titration to determine optimal concentration to minimize background

• Immunohistochemistry: May provide enhanced signal amplification but requires careful blocking to prevent endogenous biotin interference

Potential limitations:

• Background signal from endogenous biotin in tissues/cells

• Possible interference if using multiple biotin-containing reagents

• Reduced performance in tissues with high biotin content

What considerations should be made when designing experiments using biotin-conjugated ATP6V1D antibodies?

When designing experiments with biotin-conjugated ATP6V1D antibodies, researchers should consider:

• Endogenous biotin: Tissues like liver, kidney, and brain contain high levels of endogenous biotin that can cause background staining. Use appropriate blocking strategies:

  • Pre-incubate samples with avidin/streptavidin blocking solutions

  • Consider using alternative detection systems in biotin-rich tissues

• Antibody validation:

  • Confirm reactivity with your species of interest (Human, Mouse, Rat typically validated)

  • Review specificity data including Western blot bands at expected molecular weight (~28 kDa)

• Controls:

  • Include isotype controls (typically rabbit IgG for rabbit-derived antibodies)

  • Include negative controls (omission of primary antibody)

  • Consider positive control tissues with known ATP6V1D expression

• Optimization:

  • Titrate antibody concentration (starting with manufacturer's recommended dilutions)

  • Optimize incubation times and temperatures

  • Validate secondary detection reagents (streptavidin-HRP, streptavidin-fluorophore)

• Biotin interference in clinical samples:

  • Consider patient biotin supplementation history when analyzing human samples

  • Implement anti-biotin interference protocols if necessary

How can biotin interference be mitigated when using biotin-conjugated ATP6V1D antibodies in immunoassays?

Biotin interference is a significant concern in biotin-streptavidin-based immunoassays, particularly with clinical samples. Several strategies can mitigate this issue:

• Sample pre-treatment approaches:

  • Dilution of samples (effective for low to moderate biotin interference)

  • Addition of streptavidin-coated magnetic microparticles (M) to neutralize excess biotin

  • Higher concentrations of streptavidin-coated magnetic microparticles show stronger ability to resist biotin interference (up to 1000 ng/mL biotin with higher M concentrations)

• Assay modifications:

  • Use of excessive streptavidin reagents to compensate for biotin present in samples

  • Implementation of competitive binding steps to remove free biotin

  • Development of alternative detection methods that don't rely on biotin-streptavidin interactions

• Automated anti-biotin interference methods:

  • Some manufacturers have developed fully automated anti-biotin interference methods

  • These methods typically involve reagent modifications or additional neutralization steps

• Patient/sample screening:

  • Investigating biotin supplementation history

  • Testing biotin levels using ELISA or HPLC before critical assays

Research has shown that increasing the concentration of streptavidin-coated magnetic microparticles can significantly improve resistance to biotin interference, though this may affect optical signal levels .

What are the critical aspects of validating specificity for ATP6V1D biotin-conjugated antibodies?

Comprehensive validation of ATP6V1D biotin-conjugated antibodies should address:

• Epitope specificity:

  • Confirm recognition of the specific immunogen sequence (e.g., recombinant Human V-type proton ATPase subunit D protein, amino acids 1-247)

  • Test against recombinant protein fragments or full-length protein

  • Validate against negative controls (non-expressing cells/tissues)

• Cross-reactivity assessment:

  • Test against related V-ATPase subunits to ensure specificity

  • Verify reactivity across claimed species (human, mouse, rat)

  • Assess potential cross-reactivity with structurally similar proteins

• Application-specific validation:

  • Western blot: Confirm single band at expected molecular weight (~28 kDa)

  • IHC: Compare staining pattern with known ATP6V1D expression profiles

  • Flow cytometry: Compare with negative controls and blocking experiments

• Knockdown/knockout validation:

  • Test in ATP6V1D knockdown or knockout models to confirm specificity

  • Compare with alternative ATP6V1D antibodies targeting different epitopes

• Batch-to-batch consistency:

  • Implement quality control measures to ensure consistent performance

  • Compare lot performance using standardized positive controls

How can ATP6V1D antibodies be used to investigate V-ATPase complex assembly and function?

ATP6V1D antibodies are valuable tools for investigating V-ATPase complex assembly and function:

• Co-immunoprecipitation studies:

  • Use ATP6V1D antibodies to pull down intact V-ATPase complexes

  • Analyze interaction partners and complex composition under different conditions

  • Investigate the dynamic assembly/disassembly of V1 and V0 domains

• Subcellular localization:

  • Immunofluorescence studies to track ATP6V1D distribution

  • Colocalization with other V-ATPase subunits

  • Monitoring translocation between cytoplasm and membranes under different stimuli

• Functional studies:

  • Combine with pH-sensitive probes to correlate ATP6V1D localization with acidification

  • Evaluate ATP6V1D expression/localization in response to inhibitors or activators

  • Investigate its role in cilium biogenesis through protein transport and localization

• Pathological investigations:

  • Analyze expression patterns in disease models

  • Evaluate post-translational modifications using specific antibodies

  • Quantify expression levels in different tissues and disease states

• Structural biology applications:

  • Use in proximity labeling techniques to map protein interactions

  • Validate structural models of the V-ATPase complex

  • Identify critical interaction interfaces through antibody inhibition studies

What are common causes of non-specific binding when using biotin-conjugated ATP6V1D antibodies and how can they be resolved?

Non-specific binding is a common challenge when using biotin-conjugated antibodies. Key causes and solutions include:

• Endogenous biotin interference:

  • Problem: Tissues naturally contain biotin, which can bind to detection reagents

  • Solution: Use avidin/biotin blocking kits before applying biotin-conjugated antibodies

  • Alternative: Consider non-biotin detection systems for biotin-rich tissues

• Insufficient blocking:

  • Problem: Inadequate blocking leads to non-specific antibody binding

  • Solution: Optimize blocking conditions (5% BSA or 10% normal serum from the same species as the secondary antibody)

  • Alternative: Try different blocking reagents (casein, fish gelatin)

• Cross-reactivity:

  • Problem: Antibody recognizes similar epitopes on non-target proteins

  • Solution: Increase antibody dilution or use more specific antibody clones

  • Validation: Confirm specificity with knockout/knockdown controls

• Excessive antibody concentration:

  • Problem: Too much antibody increases background signal

  • Solution: Perform careful titration experiments (typically 1:500-1:2000 for WB applications)

  • Consideration: Balance signal strength with background levels

• Detection system issues:

  • Problem: Excess streptavidin-conjugate or inappropriate incubation

  • Solution: Optimize streptavidin-conjugate dilution and incubation time

  • Control: Include secondary-only controls to assess background from detection system

How should researchers interpret and validate unexpected localization patterns observed with ATP6V1D antibodies?

When encountering unexpected localization patterns with ATP6V1D antibodies, researchers should:

• Verify antibody specificity:

  • Conduct Western blot analysis to confirm single band at expected molecular weight (~28 kDa)

  • Perform peptide competition assays to confirm epitope specificity

  • Use multiple antibodies targeting different epitopes of ATP6V1D to cross-validate findings

• Consider biological contexts:

  • V-ATPase complexes can relocate under different physiological conditions

  • ATP6V1D may dissociate from the V1 complex under certain circumstances

  • Review literature for reported alternative localizations in specific cell types

• Evaluate technical factors:

  • Fixation methods can affect epitope accessibility and apparent localization

  • Permeabilization conditions influence antibody access to subcellular compartments

  • Cell/tissue preparation techniques may alter protein distribution

• Perform complementary approaches:

  • Validate with fluorescent protein-tagged ATP6V1D expression

  • Use subcellular fractionation followed by Western blotting

  • Employ super-resolution microscopy for more precise localization

• Consider functional context:

  • ATP6V1D may play roles beyond the canonical V-ATPase function

  • The protein's involvement in cilium biogenesis suggests potential non-V-ATPase localizations

  • Evaluate localization in the context of experimental treatments or disease models

What strategies can be employed to overcome limitations in detecting low abundance ATP6V1D in challenging tissue samples?

Detecting low abundance ATP6V1D in challenging samples requires specialized approaches:

• Signal amplification strategies:

  • Use biotin-streptavidin amplification systems with multiple layers

  • Employ tyramide signal amplification (TSA) for dramatically increased sensitivity

  • Consider proximity ligation assay (PLA) for enhanced specificity and sensitivity

• Sample preparation optimization:

  • Optimize antigen retrieval methods (heat-mediated retrieval is often recommended)

  • Try different fixation methods that better preserve ATP6V1D epitopes

  • Use thinner tissue sections (3-5 μm) for better antibody penetration

• Enrichment techniques:

  • Perform immunoprecipitation before analysis

  • Use subcellular fractionation to concentrate organelles where ATP6V1D is expected

  • Consider laser capture microdissection to isolate specific cell populations

• Alternative detection methods:

  • Switch from chromogenic to fluorescent detection for improved sensitivity

  • Use photomultiplier-based detection systems

  • Consider mass spectrometry-based approaches for validation

• Reducing background:

  • Implement additional blocking steps (avidin/biotin blocking, protein blocking)

  • Increase washing stringency without compromising specific signal

  • Use monovalent Fab fragments for detection in problematic tissues

• Controls and validation:

  • Include positive control tissues with known high expression

  • Use recombinant expression systems as positive controls

  • Compare results across multiple detection platforms

How are ATP6V1D antibodies being utilized to investigate the role of V-ATPase in disease models?

ATP6V1D antibodies are enabling researchers to explore V-ATPase involvement in multiple disease contexts:

• Cancer research:

  • Monitoring ATP6V1D expression in different tumor types

  • Investigating correlation between V-ATPase activity and tumor aggressiveness

  • Studying plasma membrane localization of V-ATPase in metastatic cells

  • Exploring ATP6V1D as a potential biomarker or therapeutic target

• Neurodegenerative diseases:

  • Examining ATP6V1D expression/function in models of Alzheimer's and Parkinson's diseases

  • Investigating lysosomal dysfunction related to V-ATPase activity

  • Correlating ATP6V1D levels with autophagy impairment

• Renal disorders:

  • Analyzing ATP6V1D expression in kidney tissue sections

  • Investigating the role of V-ATPase in renal tubular acidosis

  • Monitoring ATP6V1D localization in different nephron segments

• Metabolic diseases:

  • Studying ATP6V1D in models of diabetes and obesity

  • Investigating changes in V-ATPase assembly and function in metabolic stress

  • Correlating ATP6V1D with insulin signaling pathways

• Ciliopathies:

  • Examining ATP6V1D's role in cilium biogenesis

  • Investigating protein localization and transport to cilia

  • Correlating ATP6V1D dysfunction with ciliopathy phenotypes

Research techniques frequently combine immunohistochemistry, Western blotting, and colocalization studies to build comprehensive models of ATP6V1D's role in disease pathogenesis .

What are the emerging multiplex approaches for studying ATP6V1D alongside other V-ATPase subunits?

Advanced multiplex approaches are providing new insights into the coordinated function of V-ATPase subunits:

• Multiplex immunofluorescence:

  • Simultaneous detection of ATP6V1D with other V-ATPase subunits

  • Use of spectrally distinct fluorophores conjugated to antibodies against different subunits

  • Quantitative colocalization analysis to assess complex assembly

• Mass cytometry (CyTOF):

  • Metal-tagged antibodies against ATP6V1D and other V-ATPase components

  • Single-cell analysis of V-ATPase subunit expression patterns

  • Correlation with cellular phenotypes and activation states

• Spatial transcriptomics combined with protein detection:

  • In situ sequencing or spatial transcriptomics for ATP6V1D mRNA

  • Combined with immunofluorescence for protein detection

  • Correlation of transcription and translation patterns in tissue context

• Proximity-based approaches:

  • Proximity ligation assay (PLA) to detect ATP6V1D interactions with other subunits

  • FRET/BRET studies using tagged V-ATPase components

  • Split-protein complementation assays to monitor dynamic assembly

• Single molecule localization microscopy:

  • Super-resolution imaging of ATP6V1D and other V-ATPase subunits

  • Nanoscale spatial organization of V-ATPase complexes

  • Tracking of subunit dynamics in living cells

These approaches are revealing how ATP6V1D coordinates with other subunits in different physiological contexts and disease states .

What considerations should be made when designing temporal studies of V-ATPase assembly using ATP6V1D antibodies?

Temporal studies of V-ATPase assembly require careful experimental design:

• Kinetic considerations:

  • Rapid fixation methods to capture transient assembly states

  • Time-course experiments with appropriate intervals to capture dynamic processes

  • Synchronization of cells to align assembly/disassembly cycles

• Live-cell compatibility:

  • Consider membrane-permeable fluorescent-labeled Fab fragments of ATP6V1D antibodies

  • Alternative approaches using fluorescent protein-tagged ATP6V1D

  • Balance between signal strength and physiological relevance

• Stimulus-response experiments:

  • Carefully control stimuli known to affect V-ATPase assembly (glucose availability, pH changes)

  • Include appropriate vehicle controls

  • Design washout experiments to assess reversibility

• Quantification methods:

  • Develop reliable quantification of V1-V0 association states

  • Use ratiometric approaches to assess relative distribution

  • Implement automated image analysis for unbiased assessment

• Complementary biochemical approaches:

  • Combine imaging with fractionation studies at defined timepoints

  • Use crosslinking approaches to capture transient interactions

  • Correlate with functional assays of proton pumping activity

• Controls for antibody performance:

  • Ensure antibody binding is not affected by post-translational modifications that occur during assembly/disassembly

  • Validate that epitope accessibility remains consistent throughout assembly states

  • Include appropriate control proteins with stable expression/localization

These considerations help ensure that observed changes in ATP6V1D localization or interaction truly reflect biological V-ATPase assembly dynamics rather than technical artifacts .

What are the recommended quantification approaches for ATP6V1D expression analysis across different experimental platforms?

Quantification approaches should be tailored to the experimental platform while maintaining scientific rigor:

• Western blot quantification:

  • Normalization to housekeeping proteins (β-actin, GAPDH, tubulin)

  • Use of total protein normalization methods (Ponceau S, REVERT total protein stain)

  • Densitometric analysis with appropriate software (ImageJ, Image Lab)

  • Report relative expression with statistical analysis

  • Include representative blot images showing the 28 kDa band specific to ATP6V1D

• Immunohistochemistry quantification:

  • Semiquantitative scoring systems (H-score, Allred score)

  • Digital pathology approaches using automated image analysis

  • Quantification of positive cell percentage and staining intensity

  • Use of internal controls for staining normalization

  • Report both localization pattern and expression level

• Flow cytometry analysis:

  • Report median fluorescence intensity (MFI) rather than mean

  • Calculate signal-to-noise ratio relative to isotype controls

  • Use appropriate compensation when multiplexing

  • Analyze population distributions rather than just averages

  • Include representative histograms or contour plots

• ELISA/Quantitative assays:

  • Generate standard curves using recombinant ATP6V1D

  • Report concentrations in standardized units

  • Include assay detection limits and linear range

  • Analyze technical and biological replicates separately

• Cross-platform validation:

  • Confirm findings using at least two independent methods

  • Report correlation between different quantification approaches

  • Acknowledge platform-specific limitations

How should contradictory results between different ATP6V1D antibodies be interpreted and resolved?

When faced with contradictory results between different ATP6V1D antibodies:

• Epitope mapping analysis:

  • Identify the exact epitopes recognized by each antibody

  • Consider whether post-translational modifications might affect epitope accessibility

  • Evaluate whether different conformational states of ATP6V1D might affect antibody binding

  • Compare results with antibodies targeting different regions of the protein

• Validation status assessment:

  • Review the validation data for each antibody (Western blot, knockout/knockdown controls)

  • Compare specificity data across antibodies

  • Consider the validation depth for your specific application and species

• Technical optimization:

  • Test different fixation and antigen retrieval methods for each antibody

  • Optimize concentration and incubation conditions independently

  • Evaluate different detection systems to rule out conjugate-specific effects

• Biological interpretation:

  • Consider whether contradictory results reflect biological reality (alternative splicing, protein processing)

  • Evaluate tissue/cell-specific differences in ATP6V1D expression or modification

  • Assess whether different functional states of V-ATPase might affect antibody recognition

• Complementary approaches:

  • Use genetic approaches (siRNA, CRISPR) to validate antibody specificity

  • Employ mass spectrometry to confirm protein identity

  • Consider alternative detection methods (RNA analysis, reporter systems)

• Reporting contradictions:

  • Transparently report contradictory findings in publications

  • Discuss possible explanations for discrepancies

  • Provide detailed methods to allow reproducibility assessment

What statistical approaches are recommended for analyzing ATP6V1D expression across different experimental conditions or disease states?

Robust statistical analysis of ATP6V1D expression requires careful consideration:

• Experimental design considerations:

  • Power analysis to determine appropriate sample size

  • Randomization and blinding where applicable

  • Include appropriate biological and technical replicates

  • Control for batch effects and confounding variables

• Descriptive statistics:

  • Report central tendency (mean/median) and dispersion (SD/SEM/IQR)

  • Use box plots or violin plots to show distribution

  • Consider normality testing before selecting parametric/non-parametric approaches

  • Report confidence intervals alongside point estimates

• Comparative analysis:

  • For two-group comparisons: t-test (parametric) or Mann-Whitney (non-parametric)

  • For multiple groups: ANOVA with appropriate post-hoc tests (Tukey, Bonferroni)

  • For paired samples: paired t-test or Wilcoxon signed-rank test

  • For proportion data: chi-square or Fisher's exact test

• Correlation analysis:

  • Pearson correlation for linear relationships between continuous variables

  • Spearman correlation for non-parametric relationships

  • Multiple regression for controlling confounding variables

  • Report correlation coefficients with p-values and confidence intervals

• Advanced approaches:

  • For longitudinal data: mixed-effects models or repeated measures ANOVA

  • For survival analysis: Kaplan-Meier with log-rank test

  • For complex datasets: multivariate analysis (PCA, cluster analysis)

  • Consider machine learning approaches for pattern recognition in complex datasets

• Reporting standards:

  • Clearly state statistical tests used and significance thresholds

  • Address multiple comparison issues (FDR correction)

  • Report effect sizes alongside p-values

  • Make raw data available when possible

  • Use appropriate visualizations to communicate statistical findings