ATP6V1D Antibody, FITC conjugated

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

ATP6V1D (ATPase H+ Transporting V1 Subunit D) is a critical component of the vacuolar-type H+-translocating ATPase (V-ATPase), a multisubunit enzyme complex essential for cellular pH regulation. The V-ATPase complex consists of two primary assemblies: a peripheral V1 complex that hydrolyzes ATP and a membrane-integrated V0 complex that facilitates proton translocation . ATP6V1D functions within the V1 domain to support the ATP hydrolysis necessary for proton transport.

Recent research has identified ATP6V1D as a key regulator in hepatocellular carcinoma (HCC) stemness and progression, with elevated expression correlating with poor clinical outcomes in HCC patients . Mechanistically, ATP6V1D enhances cancer stemness by maintaining macroautophagic/autophagic flux through promoting lysosomal acidification and facilitating autophagosome-lysosome fusion . Beyond cancer biology, ATP6V1D may also play roles in cilium biogenesis through regulation of protein transport and localization . These diverse functions make ATP6V1D an important target for both basic science investigations and translational cancer research.

What are the key specifications of commercially available ATP6V1D antibodies conjugated to FITC?

ATP6V1D antibodies conjugated to FITC typically target the full-length protein (AA 1-247) and are available as polyclonal antibodies raised in rabbit hosts . These antibodies undergo protein G purification with purity levels exceeding 95% . The immunogens used for antibody generation are typically recombinant human V-type proton ATPase subunit D protein encompassing amino acids 1-247 . While FITC-conjugated variants are valuable for flow cytometry and immunofluorescence applications, researchers should verify reactivity with their specific target species, as human reactivity is most commonly validated .

How do ATP6V1D antibodies differ from antibodies targeting other V-ATPase subunits?

ATP6V1D antibodies specifically recognize the D subunit of the V1 complex, which plays a unique structural and functional role distinct from other V-ATPase components. Unlike antibodies against V0 complex subunits (such as ATP6V0A1) that target membrane-embedded components, ATP6V1D antibodies target a peripheral subunit involved in ATP hydrolysis rather than direct proton translocation .

Each V-ATPase subunit antibody offers distinct advantages for investigating specific aspects of V-ATPase biology. For instance, while ATP6V0A1 antibodies are useful for studying the connection between V-ATPase and immunosuppressive mechanisms in colorectal cancer , ATP6V1D antibodies are particularly valuable for investigating hepatocellular carcinoma stemness and autophagy regulation . The choice between different subunit antibodies should be guided by the specific biological process or disease mechanism under investigation.

What are the recommended storage and handling conditions for maintaining ATP6V1D antibody, FITC conjugated activity?

For optimal preservation of FITC-conjugated ATP6V1D antibodies, follow these methodological guidelines:

• Store at 2-8°C for short-term use (1-2 weeks)

• For long-term storage, aliquot and store at -20°C, avoiding repeated freeze-thaw cycles (maximum 2-3 cycles)

• Protect from prolonged light exposure due to the photosensitivity of the FITC fluorophore

• Maintain in appropriate buffer conditions (typically PBS with stabilizing proteins)

• When diluting, use buffers free of sodium azide, as this preservative can inhibit peroxidase activity in subsequent applications

Adherence to these handling protocols helps maintain conjugate stability and fluorescence intensity, which is critical for quantitative immunofluorescence applications and reproducible results across experiments.

How can ATP6V1D antibody, FITC conjugated be optimized for monitoring autophagic flux in cancer stem cell research?

Recent research has established ATP6V1D as a critical mediator of autophagic flux in hepatocellular carcinoma, particularly in maintaining cancer stem cell characteristics . When optimizing ATP6V1D antibody, FITC conjugated for investigating autophagic processes:

• Establish a dual-staining protocol combining ATP6V1D-FITC with markers of autophagosomes (LC3-II) and lysosomes (LAMP1/2) using compatible fluorophores

• Implement sequential staining approaches to minimize antibody cross-reactivity

• Include appropriate autophagy modulators as controls:

  • Rapamycin (autophagy inducer)

  • Bafilomycin A1 (V-ATPase inhibitor)

  • 3-methyladenine (3-MA, early autophagy inhibitor)

• Quantify colocalization coefficients between ATP6V1D and autophagy markers using confocal microscopy

• Validate findings using complementary approaches such as transmission electron microscopy or biochemical fractionation

This methodological approach allows for robust assessment of ATP6V1D's spatial relationship with autophagic machinery and facilitates quantitative analysis of its role in autophagosome-lysosome fusion events in cancer stem cells.

What strategies can resolve inconsistent staining patterns when using ATP6V1D antibody, FITC conjugated in different cell types?

Inconsistent staining patterns across different cell types may reflect biological variations in ATP6V1D expression, subcellular localization, or technical limitations. To address this methodological challenge:

• Validate antibody specificity using positive and negative controls:

  • ATP6V1D knockdown or knockout cells as negative controls

  • Cells with confirmed high ATP6V1D expression as positive controls

  • Western blot validation showing a single band at approximately 28 kDa

• Optimize fixation protocols based on cellular compartmentalization:

  • For peripheral V1 complex: 4% paraformaldehyde (10-15 minutes)

  • For membrane-associated complexes: Methanol fixation (-20°C, 10 minutes)

  • For detecting both pools: Sequential fixation with paraformaldehyde followed by methanol

• Implement cell type-specific permeabilization:

  • Epithelial cells: 0.1-0.2% Triton X-100

  • Hepatocytes: 0.05% saponin

  • Neurons: 0.1% Tween-20

• Adjust antibody concentration based on expression levels:

  • High-expressing cells: 1:200-1:500 dilution

  • Low-expressing cells: 1:50-1:100 dilution

• Extend incubation times for cells with complex matrices:

  • Standard protocol: 1-2 hours at room temperature

  • Modified protocol: Overnight at 4°C with gentle agitation

These adjustments account for cell-specific variables that influence ATP6V1D detection and help establish consistent staining protocols across diverse experimental systems.

How can ATP6V1D antibody, FITC conjugated be used to investigate the relationship between V-ATPase function and cancer therapeutic resistance?

Emerging evidence suggests V-ATPase components including ATP6V1D contribute to therapeutic resistance in multiple cancers. To investigate this relationship using FITC-conjugated ATP6V1D antibodies:

• Design flow cytometry panels to correlate ATP6V1D expression with known resistance markers:

  • ATP6V1D-FITC

  • Drug resistance proteins (e.g., P-glycoprotein-PE)

  • Stemness markers (e.g., CD44-APC)

• Implement sequential treatment protocols:

  • Pre-treatment measurement of ATP6V1D levels

  • Exposure to therapeutic agents

  • Post-treatment assessment of ATP6V1D expression and localization

• Establish cell sorting strategies to isolate ATP6V1D-high and ATP6V1D-low populations for:

  • Drug sensitivity testing

  • Transcriptomic profiling

  • Functional assessment of lysosomal acidification

• Utilize combination treatments targeting V-ATPase function:

  • Standard chemotherapeutics + low-dose bafilomycin A1

  • Monitor treatment response using ATP6V1D-FITC as a biomarker

• Correlate ATP6V1D expression with clinical outcomes:

  • Patient-derived xenograft models

  • Tissue microarray analysis from resistant vs. responsive tumors

This methodological framework facilitates mechanistic investigation of how ATP6V1D expression and V-ATPase function contribute to therapeutic resistance, potentially identifying new combinatorial approaches for cancer treatment.

What are the optimal fixation and permeabilization protocols for ATP6V1D immunofluorescence using FITC-conjugated antibodies?

The detection of ATP6V1D using FITC-conjugated antibodies requires careful optimization of fixation and permeabilization steps to maintain both epitope integrity and fluorophore activity. Based on the subcellular localization of ATP6V1D in both cytosolic V1 complexes and membrane-associated V-ATPase assemblies, a comprehensive protocol would include:

Optimized Fixation Protocol:

• Wash cells twice with pre-warmed PBS (37°C)

• Fix with 4% paraformaldehyde in PBS for 15 minutes at room temperature

• If detecting membrane-associated pools, perform optional post-fixation with ice-cold methanol (-20°C) for 5 minutes

• Wash three times with PBS (5 minutes each)

Permeabilization Options:

Following permeabilization, implement a blocking step with 5% normal serum from the same species as the secondary antibody (if using indirect detection) or 5% BSA for 30-60 minutes before proceeding with the ATP6V1D-FITC antibody incubation.

How can potential cross-reactivity issues with ATP6V1D antibody, FITC conjugated be identified and mitigated?

Cross-reactivity remains a significant challenge when working with antibodies against V-ATPase components due to structural similarities between subunits. To identify and address potential cross-reactivity:

• Validation approaches:

  • Perform Western blot analysis to confirm a single band at the expected molecular weight (~28 kDa for ATP6V1D)

  • Utilize ATP6V1D knockout or knockdown models as negative controls

  • Compare staining patterns with multiple ATP6V1D antibodies recognizing different epitopes

• Pre-absorption controls:

  • Pre-incubate the ATP6V1D-FITC antibody with recombinant ATP6V1D protein

  • Apply to parallel samples to identify non-specific binding

  • Non-specific signal should disappear in pre-absorbed samples

• Species-specific considerations:

  • Human ATP6V1D shares high homology with mouse and rat orthologs

  • Verify species cross-reactivity through sequence alignment of the immunogen region

  • Incorporate species-specific positive controls

• Mitigating strategies:

  • Increase washing steps (5-6 washes instead of standard 3)

  • Use higher dilutions of antibody (starting at 1:200 and titrating)

  • Add 0.05% Tween-20 to wash buffers to reduce non-specific binding

  • Implement background reduction agents like 0.1-0.3M glycine

These approaches help establish antibody specificity and minimize false positive results that could confound interpretation of ATP6V1D localization and expression studies.

What are the recommended protocols for multiplexing ATP6V1D-FITC antibody with other fluorescent markers for colocalization studies?

Multiplexing ATP6V1D-FITC antibody with other markers enables comprehensive analysis of ATP6V1D's relationship with cellular structures and interaction partners. For successful multiplexing:

• Fluorophore selection to minimize spectral overlap:

  • FITC (ATP6V1D): Excitation 495nm / Emission 519nm

  • Compatible partners:

    • TRITC/RhodamineRed: Ex 557nm / Em 576nm

    • Cy5: Ex 650nm / Em 670nm

    • Pacific Blue: Ex 410nm / Em 455nm

• Sequential staining protocol for multi-antibody labeling:

  • Begin with the lowest abundance target

  • Apply primary antibodies sequentially if using indirect detection

  • For directly conjugated antibodies (like ATP6V1D-FITC), apply in order of increasing fluorophore brightness

• Recommended combinations for V-ATPase biology:

  Target	Fluorophore	Purpose	Dilution Range
  ATP6V1D	FITC	V1 complex	1:100-1:200
  ATP6V0A	TRITC	V0 complex	1:100-1:200
  LAMP1	Cy5	Lysosomes	1:100-1:500
  LC3B	Pacific Blue	Autophagosomes	1:100-1:500

• Controls for accurate colocalization assessment:

  • Single-color controls for spectral compensation

  • Fluorescence minus one (FMO) controls

  • Biological controls (e.g., bafilomycin A1 treatment to disrupt V-ATPase assembly)

• Image acquisition and analysis considerations:

  • Use sequential scanning to minimize bleed-through

  • Apply deconvolution algorithms for improved signal-to-noise ratio

  • Quantify colocalization using Pearson's or Mander's coefficients

This methodological approach facilitates detailed investigation of ATP6V1D's spatial relationships with other V-ATPase components and associated cellular structures.

How can ATP6V1D antibody, FITC conjugated be used to investigate the role of V-ATPase in cancer stemness and progression?

Recent research has established ATP6V1D as a key driver of hepatocellular carcinoma stemness and progression . To investigate this relationship using ATP6V1D-FITC antibodies:

• Flow cytometry protocol for cancer stem cell identification:

  • Prepare single-cell suspensions from tumor samples or cell lines

  • Co-stain with ATP6V1D-FITC and established cancer stem cell markers (CD44, CD133, EpCAM)

  • Gate on marker-positive populations and quantify ATP6V1D expression levels

  • Sort ATP6V1D-high and ATP6V1D-low populations for functional assays

• Sphere formation assay correlation:

  • Isolate cells based on ATP6V1D expression levels

  • Plate equal numbers in low-attachment conditions with stem cell media

  • Quantify sphere formation efficiency and size

  • Re-analyze ATP6V1D expression in formed spheres versus adherent cultures

• Patient sample analysis workflow:

  • Process tissue samples with enzymatic digestion (collagenase/dispase)

  • Implement ATP6V1D-FITC staining in multiparameter flow panels

  • Correlate ATP6V1D expression with:

    • Clinical outcomes

    • Treatment response

    • Recurrence rates

• Mechanism investigation:

  • Monitor ATP6V1D expression during cell differentiation using time-course analysis

  • Assess ATP6V1D localization changes during epithelial-mesenchymal transition

  • Quantify the relationship between ATP6V1D levels and lysosomal acidification using LysoTracker co-staining

This comprehensive approach enables detailed characterization of ATP6V1D's role in maintaining cancer stem cell properties and facilitates the identification of potential therapeutic vulnerabilities.

What are the key considerations when optimizing ATP6V1D-FITC antibody for flow cytometry applications?

Flow cytometry with ATP6V1D-FITC antibodies requires careful optimization due to the predominantly intracellular localization of ATP6V1D. Follow these methodological considerations:

• Cell preparation protocol:

  • Fix cells with 2% paraformaldehyde (15 minutes, room temperature)

  • Permeabilize with 0.1% saponin in PBS with 0.5% BSA (20 minutes, room temperature)

  • Maintain saponin in all subsequent buffers to preserve permeabilization

• Antibody titration matrix:

  Antibody Dilution	Cell Number	Incubation Time	Temperature
  1:50	1×10^6	30 min	4°C
  1:100	1×10^6	30 min	4°C
  1:200	1×10^6	30 min	4°C
  1:500	1×10^6	30 min	4°C

• Critical controls:

  • Unstained cells for autofluorescence assessment

  • Isotype control conjugated to FITC

  • FMO (Fluorescence Minus One) controls for multiparameter panels

  • ATP6V1D knockdown cells as biological negative controls

• Optimization for co-staining with stem cell markers:

  • Use Fc receptor blocking (15 minutes) before antibody addition

  • Apply surface marker antibodies before fixation/permeabilization

  • After permeabilization, add ATP6V1D-FITC antibody

  • Wash thoroughly (3-4 times) to remove unbound antibody

• Instrument setup considerations:

  • Use FITC single-stained controls for proper compensation

  • Adjust voltage to position negative population appropriately (first decade)

  • Acquire sufficient events (minimum 30,000) for reliable statistics

  • Apply doublet discrimination to ensure single-cell analysis

These optimization steps ensure reliable and quantitative assessment of ATP6V1D expression in diverse cell populations, facilitating correlation with cellular phenotypes and disease states.

What experimental design is recommended for investigating ATP6V1D's role in autophagosome-lysosome fusion using FITC-conjugated antibodies?

To investigate ATP6V1D's role in autophagosome-lysosome fusion using fluorescence microscopy:

• Experimental groups design:

  • Control cells (baseline autophagy)

  • Starvation-induced autophagy (EBSS medium, 2-4 hours)

  • Bafilomycin A1 treatment (100 nM, 4-6 hours) to inhibit V-ATPase

  • Chloroquine treatment (50 μM, 4-6 hours) to raise lysosomal pH

  • ATP6V1D knockdown/knockout cells

• Triple-staining protocol:

  • ATP6V1D-FITC: Detection of V-ATPase D subunit

  • Anti-LC3B-TRITC: Autophagosome marker

  • Anti-LAMP1-Cy5: Lysosome marker

• Time-course imaging setup:

  Timepoint	Purpose	Analysis Focus
  0h	Baseline	Resting distribution
  2h	Early autophagy	Autophagosome formation
  4h	Peak fusion	Autophagolysosome formation
  8h	Late autophagy	Clearance dynamics

• Quantitative colocalization analysis:

  • Measure Pearson's correlation coefficient between:

    • ATP6V1D and LC3B

    • ATP6V1D and LAMP1

    • Triple colocalization (ATP6V1D/LC3B/LAMP1)

  • Track changes in colocalization metrics across experimental conditions

  • Correlate with functional autophagy readouts (p62 degradation, LC3-II/I ratio)

• Advanced imaging approaches:

  • Live-cell imaging using ATP6V1D-GFP fusion proteins with RFP-LC3 and LAMP1-BFP

  • Super-resolution microscopy (STED or STORM) for detailed interaction analysis

  • FRET-based approaches to measure direct protein-protein interactions

This experimental design facilitates comprehensive analysis of ATP6V1D's spatial and temporal relationships during autophagy progression, providing mechanistic insights into its role in autophagosome-lysosome fusion.

What are the most promising research directions for ATP6V1D antibody applications in cancer and neurodegenerative disease research?

The ATP6V1D antibody, particularly in FITC-conjugated format, offers significant potential for advancing understanding in several critical research areas:

• Cancer research applications:

  • Biomarker development for identifying therapy-resistant cancer stem cells

  • Monitoring autophagy modulation during combined treatment approaches

  • Investigating the relationship between lysosomal acidification and immune evasion

  • Exploring ATP6V1D as a predictive marker for response to autophagy inhibitors

• Neurodegenerative disease investigations:

  • Assessing V-ATPase dysfunction in lysosomal storage disorders

  • Tracking ATP6V1D expression changes in Alzheimer's and Parkinson's disease models

  • Investigating autophagy defects in neurodegeneration using ATP6V1D as a marker

  • Correlating ATP6V1D function with protein aggregation clearance mechanisms

• Emerging therapeutic strategies:

  • Monitoring on-target engagement of V-ATPase inhibitors

  • Validating ATP6V1D as a druggable target for autophagy modulation

  • Developing ATP6V1D expression as a companion diagnostic for autophagy-targeted therapies

  • Exploring combination strategies targeting both ATP6V1D function and downstream pathways

The continued refinement of ATP6V1D antibody applications will facilitate deeper mechanistic understanding of V-ATPase biology in disease contexts, potentially leading to novel therapeutic approaches and diagnostic tools that leverage this critical cellular machinery.