ATP6V0D1 Antibody

What is ATP6V0D1 and why is it important in cellular physiology?

ATP6V0D1 (V-type proton ATPase subunit d 1) is a subunit of the V0 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, providing the energy required for transport processes in the vacuolar system . In aerobic conditions, ATP6V0D1 is involved in intracellular iron homeostasis, triggering the activity of Fe(2+) prolyl hydroxylase (PHD) enzymes, leading to HIF1A hydroxylation and subsequent proteasomal degradation . Additionally, it may play a role in cilium biogenesis through regulation of protein transport and localization .

What are the key specifications of commercial ATP6V0D1 antibodies?

ATP6V0D1 antibodies are available in multiple formats with varied specifications:

What are the differences between monoclonal and polyclonal ATP6V0D1 antibodies in experimental applications?

The choice between monoclonal and polyclonal ATP6V0D1 antibodies depends on experimental requirements:

Monoclonal antibodies (e.g., Abcam ab56441 [2G12] or ab202897 [EPR18320]) recognize a single epitope, providing superior specificity and consistent reproducibility across experiments . They are particularly valuable for applications requiring precise epitope recognition, such as distinguishing between protein isoforms or specific post-translational modifications.

For critical applications, using both antibody types in parallel can provide complementary data, enhancing result reliability and interpretation.

How should Western blot protocols be optimized for ATP6V0D1 detection?

For optimal Western blot detection of ATP6V0D1:

• Sample preparation:

  • Extract proteins from tissues or cell lines with confirmed ATP6V0D1 expression (HEK-293, HeLa, human placenta, mouse kidney, mouse testis)

  • Include protease inhibitors to prevent degradation

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution around 37-41 kDa

  • Ensure complete transfer to PVDF or nitrocellulose membranes

• Antibody incubation:

  • Primary antibody dilutions vary by manufacturer:

    • Proteintech 18274-1-AP: 1:2000-1:10000

    • Boster A10658: 1:500-1:2000

    • Fisher Scientific/Novus: 1:500-1:2000

  • Optimize blocking conditions (5% non-fat milk or BSA in TBST)

  • Incubate primary antibody overnight at 4°C for maximum sensitivity

• Detection optimization:

  • Use HRP-conjugated secondary antibodies at 1:5000-1:10000

  • For low expression samples, consider enhanced chemiluminescence substrates

  • For quantitative analysis, ensure signal is within linear detection range

• Controls:

  • Include positive controls (HeLa or HEK-293 lysates)

  • Consider lysates from ATP6V0D1 knockdown/knockout cells as negative controls

Titrate antibody concentrations in each system to determine optimal conditions for your specific samples .

What are the critical considerations for immunohistochemical detection of ATP6V0D1?

For successful immunohistochemical detection of ATP6V0D1:

• Tissue preparation and antigen retrieval:

  • For FFPE sections, ATP6V0D1 antibodies work well with both citrate buffer (pH 6.0) and TE buffer (pH 9.0) antigen retrieval methods

  • Optimal section thickness is typically 3-5 μm

• Antibody selection and dilution:

  • For IHC-P applications, recommended dilutions range from 1:50-1:500

  • Both rabbit polyclonal (Proteintech) and mouse monoclonal antibodies (Abcam) have been validated for IHC

• Visualization systems:

  • DAB-based detection systems provide excellent sensitivity

  • For multiplex staining, consider fluorescent secondary antibodies

• Validated positive control tissues:

  • Human kidney tissue shows reliable ATP6V0D1 expression

  • Mouse kidney tissue also demonstrates consistent staining

• Specific considerations:

  • Perform careful titration experiments to minimize background

  • Include appropriate negative controls (omitting primary antibody)

  • Consider automated staining platforms for enhanced reproducibility

• Signal interpretation:

  • ATP6V0D1 typically shows cytoplasmic and membrane-associated patterns

  • Compare staining patterns with subcellular localization data from other methods

When analyzing tissues with variable ATP6V0D1 expression, optimize exposure times and detection sensitivity based on control tissues with known expression levels.

What methodologies enable successful co-immunoprecipitation of ATP6V0D1 and its interaction partners?

For effective co-immunoprecipitation of ATP6V0D1 and its binding partners:

• Lysis buffer selection:

  • Use mild non-denaturing buffers (e.g., NP-40 or Triton X-100 based) to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors to maintain complex integrity

• Pre-clearing step:

  • Pre-clear lysates with appropriate control IgG and protein A/G beads

  • This reduces non-specific binding and improves signal-to-noise ratio

• Antibody selection:

  • Use 0.5-4.0 μg of ATP6V0D1 antibody per 1.0-3.0 mg of total protein lysate

  • Both polyclonal (Proteintech 18274-1-AP) and recombinant monoclonal antibodies (Abcam ab202897) have been validated for IP

• Optimized protocol:

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

  • Add protein A/G beads and continue incubation for 2-4 hours

  • Perform stringent washes while preserving specific interactions

  • Elute complexes with sample buffer or gentle elution methods

• Interaction verification:

  • Analyze eluates by Western blot for ATP6V0D1 and suspected interaction partners

  • For comprehensive analysis, consider mass spectrometry identification of co-precipitated proteins

• Controls:

  • Include IgG control immunoprecipitations

  • Consider reverse immunoprecipitation with antibodies against suspected interaction partners

Mouse testis tissue has been validated for successful immunoprecipitation of ATP6V0D1 , making it an excellent positive control for establishing the methodology.

How can ATP6V0D1 antibodies be employed to study V-ATPase complex assembly and function?

ATP6V0D1 antibodies provide valuable tools for investigating V-ATPase assembly and function:

• Complex assembly analysis:

  • Immunoprecipitate ATP6V0D1 and probe for other V-ATPase subunits (V1 and V0 components)

  • Perform native PAGE followed by Western blotting to preserve intact complexes

  • Compare complex assembly under different physiological or pathological conditions

• Subcellular localization studies:

  • Use fluorescently labeled ATP6V0D1 antibodies (e.g., CoraLite® Plus 488 conjugate)

  • Co-stain with organelle markers to track V-ATPase distribution

  • Employ super-resolution microscopy for nanoscale localization analysis

• Functional assessments:

  • Correlate ATP6V0D1 expression/localization with V-ATPase activity using lysosomal pH sensors

  • Analyze the effects of V-ATPase inhibitors on ATP6V0D1 distribution

  • Study how ATP6V0D1 knockdown affects V-ATPase assembly and function

• Post-translational modifications:

  • Immunoprecipitate ATP6V0D1 under various conditions

  • Probe for modifications that might regulate complex assembly (phosphorylation, ubiquitination)

  • Correlate modifications with functional changes

• Dynamic studies:

  • Use live-cell imaging with membrane-permeable fluorescent nanobodies

  • Track ATP6V0D1-containing complexes in real-time

  • Correlate dynamics with cellular functions like autophagy or endocytosis

These approaches can reveal how ATP6V0D1 contributes to V-ATPase assembly, localization, and function across different cellular compartments and physiological states.

What insights can ATP6V0D1 antibodies provide in studies of autophagy and lysosomal function?

ATP6V0D1 antibodies offer powerful tools for investigating autophagy and lysosomal biology:

• Autophagy flux assessment:

  • Monitor ATP6V0D1 expression and localization during autophagy induction

  • Correlate changes with autophagy markers (LC3-II, p62/SQSTM1)

  • Investigate how V-ATPase inhibitors affect autophagosome-lysosome fusion

• Lysosomal acidification studies:

  • Combine ATP6V0D1 immunostaining with LysoTracker or LysoSensor probes

  • Correlate ATP6V0D1 expression/localization with lysosomal pH

  • Assess acidification defects in disease models or after genetic manipulation

• Protein degradation pathways:

  • Examine ATP6V0D1 distribution in relation to endocytic and autophagic markers

  • Investigate the role of V-ATPase in different degradative pathways

  • Analyze how ATP6V0D1 knockdown affects protein turnover

• Stress response mechanisms:

  • Study ATP6V0D1 dynamics during nutrient deprivation, oxidative stress, or ER stress

  • Investigate connections between V-ATPase activity and AMPK or mTORC1 signaling

  • Explore how V-ATPase contributes to cellular adaptation to stress

• Disease models:

  • Compare ATP6V0D1 expression and localization in models of lysosomal storage disorders

  • Analyze V-ATPase function in neurodegenerative diseases associated with autophagy defects

  • Investigate potential therapeutic strategies targeting V-ATPase activity

The ATP6V0D1 antibody validated in human and mouse kidney tissues is particularly valuable for these studies, as kidney cells are rich in lysosomes and display prominent autophagy activity.

How can ATP6V0D1 antibodies contribute to understanding the role of V-ATPase in cancer biology?

ATP6V0D1 antibodies provide crucial tools for exploring V-ATPase's role in cancer pathophysiology:

• Expression profiling across cancer types:

  • Perform immunohistochemistry on tissue microarrays from various tumors

  • Quantify ATP6V0D1 expression levels and correlate with clinical parameters

  • Compare expression between tumor tissue and adjacent normal tissue

• Subcellular relocalization analysis:

  • Investigate ATP6V0D1 redistribution to plasma membrane in invasive cancers

  • Correlate plasma membrane localization with extracellular acidification

  • Study how ATP6V0D1 localization affects tumor microenvironment

• Drug resistance mechanisms:

  • Compare ATP6V0D1 expression in drug-sensitive versus resistant cell lines

  • Analyze if chemotherapy treatment alters V-ATPase distribution

  • Explore how V-ATPase inhibition modulates therapeutic response

• Invasion and metastasis:

  • Examine ATP6V0D1 expression at invasive tumor fronts

  • Correlate expression with markers of epithelial-mesenchymal transition

  • Study how ATP6V0D1 knockdown affects migration and invasion in vitro

• Metabolic adaptation:

  • Investigate relationships between ATP6V0D1 expression and metabolic markers

  • Study how V-ATPase activity supports cancer cell metabolism

  • Examine ATP6V0D1 regulation under hypoxic conditions

• Therapeutic targeting:

  • Use ATP6V0D1 antibodies to monitor the effects of V-ATPase inhibitors

  • Identify optimal dosing schedules through time-course studies

  • Develop combination strategies targeting V-ATPase and related pathways

Since ATP6V0D1 antibodies have been validated in multiple cell lines including HeLa , they provide reliable tools for cancer-focused investigations.

What are common challenges in ATP6V0D1 antibody applications and how can they be resolved?

Addressing common technical issues with ATP6V0D1 antibody applications:

• Weak or absent Western blot signal:

  • Increase antibody concentration within recommended range (1:500-1:10000)

  • Extend primary antibody incubation time to overnight at 4°C

  • Optimize protein extraction method to ensure ATP6V0D1 solubilization

  • Include positive controls (HeLa, HEK-293, mouse kidney tissue)

  • Verify protein transfer efficiency with reversible staining

• High background in immunohistochemistry/immunofluorescence:

  • Optimize blocking conditions (increase blocking time or concentration)

  • Dilute primary antibody further (start at 1:500 and adjust as needed)

  • Increase washing duration and number of washes

  • For IHC, compare citrate buffer (pH 6.0) versus TE buffer (pH 9.0) antigen retrieval

  • Consider using monoclonal antibodies for higher specificity

• Poor immunoprecipitation efficiency:

  • Adjust antibody amount to 0.5-4.0 μg per 1.0-3.0 mg of lysate

  • Optimize lysis buffer composition to better preserve protein-protein interactions

  • Extend antibody-lysate incubation time to enhance antigen capture

  • Consider using different antibody clones that recognize distinct epitopes

• Inconsistent staining patterns:

  • Standardize fixation time and conditions

  • Maintain consistent antibody lots when possible

  • Include internal controls within each experiment

  • For fluorescent detection, minimize exposure to light to prevent photobleaching

• Non-specific bands in Western blot:

  • Increase washing stringency (more/longer washes, higher detergent concentration)

  • Use freshly prepared buffers and reagents

  • Consider using gradient gels for better resolution around 37-41 kDa

  • Block with 5% BSA instead of milk for phospho-specific detection

How can researchers validate the specificity of ATP6V0D1 antibodies?

Rigorous validation of ATP6V0D1 antibody specificity requires multiple complementary approaches:

• Genetic validation:

  • Test antibody in ATP6V0D1 knockdown/knockout systems

  • Compare signal reduction/elimination with target depletion

  • Several publications have used ATP6V0D1 KD/KO systems for validation

• Multiple antibody comparison:

  • Use different antibodies targeting distinct ATP6V0D1 epitopes

  • Compare staining patterns across applications

  • Consistent results with different antibodies increase confidence in specificity

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Verify signal reduction in Western blot or immunostaining

  • This confirms epitope-specific binding

• Recombinant protein controls:

  • Test antibody against recombinant ATP6V0D1

  • Compare with endogenous protein detection

  • Verify correct molecular weight recognition (37-41 kDa)

• Mass spectrometry validation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Confirm ATP6V0D1 enrichment in immunoprecipitated samples

  • Identify potential cross-reactive proteins

• Species cross-reactivity testing:

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

  • Ensure consistent staining patterns in conserved tissues

  • This is particularly important for comparative studies

A combination of these approaches provides comprehensive validation and increases confidence in experimental results.

What considerations are important when selecting between different commercial ATP6V0D1 antibodies?

Strategic selection of ATP6V0D1 antibodies should consider:

• Application compatibility:

  • Specific applications require differently validated antibodies:

    • For Western blot: Most antibodies are validated (1:500-1:10000 dilution range)

    • For IP: Select antibodies specifically validated for immunoprecipitation

    • For IHC: Consider antibodies validated on FFPE tissues

    • For IF: Directly conjugated antibodies offer advantages for co-localization studies

• Host species considerations:

  • For multi-color immunofluorescence, select antibodies from different host species

  • Consider secondary antibody availability and specificity

  • For in vivo applications, evaluate potential immunogenicity

• Clonality trade-offs:

  • Polyclonal antibodies: Higher sensitivity but potentially more background

  • Monoclonal antibodies: Superior specificity but may be epitope-restricted

  • Recombinant monoclonal antibodies: Consistent reproducibility between lots

• Epitope location:

  • N-terminal vs. C-terminal targeting affects detection of truncated variants

  • Internal epitopes may be masked by protein-protein interactions

  • Compare immunogen sequences across manufacturers

• Validation depth:

  • Evaluate the extent of validation data provided by manufacturers

  • Consider antibodies with publication history in your application of interest

  • Antibodies validated by multiple techniques offer higher confidence

• Special formats:

  • Consider conjugated antibodies for direct detection

  • Evaluate BSA-free formulations for certain applications

  • Assess azide-free options for functional assays

Selecting the optimal ATP6V0D1 antibody requires balancing these considerations against your specific experimental requirements and technical constraints.

How are ATP6V0D1 antibodies being utilized in studies of neurodegenerative diseases?

ATP6V0D1 antibodies are providing valuable insights into neurodegenerative disease mechanisms:

• Lysosomal dysfunction analysis:

  • Compare ATP6V0D1 expression and localization in affected versus unaffected brain regions

  • Correlate with markers of lysosomal function (LAMP1, cathepsins)

  • Investigate relationships between V-ATPase activity and protein aggregation

• Cell type-specific investigations:

  • Combine ATP6V0D1 immunostaining with neuronal, glial, or microglial markers

  • Analyze cell type-specific changes in V-ATPase distribution

  • Assess differential vulnerability to lysosomal dysfunction

• Disease model applications:

  • Study ATP6V0D1 dynamics in animal models of Alzheimer's, Parkinson's, or ALS

  • Compare findings with human post-mortem tissue

  • Investigate if restoring proper V-ATPase function ameliorates disease phenotypes

• Therapeutic development:

  • Use ATP6V0D1 antibodies to monitor effects of compounds targeting lysosomal function

  • Assess V-ATPase assembly/function in response to potential therapeutics

  • Develop biomarkers for lysosomal dysfunction in neurodegenerative diseases

• Technical considerations for neural tissue:

  • Optimize antigen retrieval for brain tissue (both TE buffer pH 9.0 and citrate buffer pH 6.0 have been validated)

  • Account for lipid-rich environment when extracting proteins

  • Address autofluorescence issues in aged brain tissue

The validated reactivity of ATP6V0D1 antibodies across human, mouse, and rat samples makes them particularly valuable for translational neuroscience research.

What methodological approaches enable investigation of ATP6V0D1 post-translational modifications?

Investigating ATP6V0D1 post-translational modifications requires systematic approaches:

• Phosphorylation analysis:

  • Immunoprecipitate ATP6V0D1 using validated antibodies (0.5-4.0 μg per sample)

  • Probe with phospho-specific antibodies (anti-phospho-serine/threonine/tyrosine)

  • Perform phosphatase treatment controls to confirm specificity

  • Compare phosphorylation status across experimental conditions

• Ubiquitination studies:

  • Conduct immunoprecipitation under denaturing conditions to disrupt protein interactions

  • Probe with anti-ubiquitin antibodies

  • Use proteasome inhibitors to enhance detection of ubiquitinated species

  • Distinguish between mono- and poly-ubiquitination patterns

• Mass spectrometry-based approaches:

  • Perform large-scale immunoprecipitation of ATP6V0D1

  • Digest proteins and analyze by LC-MS/MS

  • Use phospho-enrichment techniques to enhance detection of low-abundance modifications

  • Map identified modifications to ATP6V0D1 sequence and structural domains

• Functional correlation:

  • Investigate how modifications affect V-ATPase assembly using co-immunoprecipitation

  • Correlate modification status with subcellular localization using immunofluorescence

  • Assess impact on protein stability through cycloheximide chase experiments

• Stimulus-dependent modification:

  • Monitor ATP6V0D1 modifications in response to nutrient availability, pH changes, or stress

  • Correlate with V-ATPase activity measurements

  • Identify regulatory kinases/phosphatases through inhibitor studies

These methodologies enable characterization of the complex post-translational regulation of ATP6V0D1, providing insights into V-ATPase regulation under various physiological and pathological conditions.

How can super-resolution microscopy enhance ATP6V0D1 localization studies?

Super-resolution microscopy combined with ATP6V0D1 antibodies offers unprecedented insights into V-ATPase organization:

• Sample preparation optimization:

  • Use high-affinity ATP6V0D1 antibodies validated for immunofluorescence (1:50-1:500 dilution)

  • For direct STORM, conjugate antibodies with appropriate photoswitchable fluorophores

  • For STED, select fluorophores with optimal depletion characteristics

  • Consider directly conjugated antibodies like CoraLite® Plus 488 ATP6V0D1

• Multi-color super-resolution strategies:

  • Combine ATP6V0D1 staining with markers for subcellular compartments

  • Use sequential imaging to minimize chromatic aberrations

  • Apply drift correction algorithms for precise alignment

• V-ATPase complex visualization:

  • Image ATP6V0D1 alongside other V-ATPase subunits at nanoscale resolution

  • Measure precise distances between components (10-20 nm precision)

  • Analyze clustering patterns and complex assembly

• Specialized applications:

  • Expansion microscopy: Physically expand samples to achieve super-resolution with standard confocal microscopy

  • DNA-PAINT: Use transient binding of labeled DNA oligos for ultra-high precision localization

  • Correlative light-electron microscopy: Combine super-resolution with ultrastructural analysis

• Quantitative analysis:

  • Implement cluster analysis algorithms to identify ATP6V0D1 distribution patterns

  • Measure co-localization with nanometer precision using coordinate-based methods

  • Track changes in spatial organization under different experimental conditions

These advanced imaging approaches reveal ATP6V0D1 distribution and organization at previously inaccessible scales, providing new insights into V-ATPase complex assembly and regulation.