ATP6V1H Antibody

What is ATP6V1H and why is it important in cellular function?

ATP6V1H is a regulatory subunit H of the V1 domain of vacuolar(H+)-ATPase (V-ATPase), a multisubunit enzyme complex. This complex consists of two main components: a peripheral V1 domain that hydrolyzes ATP and a membrane-integrated V0 domain that translocates protons . ATP6V1H is essential for the V-ATPase activity but interestingly is not required for the assembly of the complex itself .

The V-ATPase complex plays critical roles in:

• Acidifying intracellular compartments and maintaining pH homeostasis

• Supporting receptor-mediated endocytosis and intracellular trafficking processes

• Facilitating protein degradation pathways

• Contributing to clathrin-mediated endocytosis, specifically in endosome formation

Recent research has also implicated ATP6V1H in metabolic regulation, particularly in pancreatic islet function, with potential connections to type 2 diabetes pathogenesis .

What types of ATP6V1H antibodies are available for research applications?

Several types of ATP6V1H antibodies are available for research applications:

These antibodies provide researchers with multiple options depending on their experimental requirements and target species .

How does ATP6V1H function within the V-ATPase complex?

ATP6V1H serves as a crucial regulatory subunit within the V-ATPase complex with several specific functions:

• It acts as an essential component for the catalytic activity of V-ATPase, though it is not required for complex assembly .

• The subunit participates in regulating the coupling between ATP hydrolysis in the V1 domain and proton transport through the V0 domain.

• It is involved in the endocytosis pathway mediated by clathrin-coated pits, specifically required for the formation of endosomes .

• ATP6V1H has been identified as a Nef-binding protein (NBP1), suggesting potential involvement in additional protein-protein interactions .

The unique regulatory role of ATP6V1H makes it distinct from structural subunits, positioning it as a potential target for studying V-ATPase regulation in various cellular contexts and disease models .

What are the optimal conditions for using ATP6V1H antibodies in Western blot applications?

For optimal Western blot detection of ATP6V1H, the following methodological considerations are important:

• Antibody Selection: Choose an antibody validated for Western blot applications. For example, antibody ab187706 has been validated for WB applications with human and mouse samples . The H00051606-D01P antibody is also suitable, particularly when detecting overexpressed ATP6V1H in transfected cells .

• Antibody Dilution:

  • For ab187706: Follow manufacturer recommendations for optimal dilution

  • For HPA023421: Use at 0.04-0.4 μg/mL

  • For H00051606-D01P: Experimentally determine optimal dilutions

• Sample Preparation: Since ATP6V1H is part of a multisubunit complex, proper sample preparation is crucial. Use lysis buffers that effectively solubilize membrane-associated proteins without disrupting antibody epitopes.

• Expected Molecular Weight: ATP6V1H appears at approximately 55.90 KDa on Western blots as observed with the H00051606-D01P antibody when detecting the protein in transfected 293T cell lines .

• Controls: Include non-transfected lysates as negative controls when working with overexpression systems, as demonstrated in the validation of H00051606-D01P antibody .

Following these guidelines will help ensure specific detection of ATP6V1H in Western blot applications.

What are the recommended protocols for immunohistochemistry detection of ATP6V1H?

For successful immunohistochemical detection of ATP6V1H, researchers should consider the following protocol elements:

• Antibody Selection: The HPA023421 antibody has been extensively validated for immunohistochemistry through the Human Protein Atlas project, making it a strong choice for IHC applications . The ab254685 antibody is also validated for IHC-P (paraffin-embedded tissues) .

• Dilution Range: For HPA023421, a dilution range of 1:50-1:200 is recommended for immunohistochemistry applications .

• Validation Strategy: The Human Protein Atlas project has tested HPA023421 on:

  • IHC tissue arrays of 44 normal human tissues

  • 20 of the most common cancer type tissues

  • Protein arrays of 364 human recombinant protein fragments

• Tissue Fixation: Standard formalin fixation and paraffin embedding protocols are compatible with these antibodies.

• Antigen Retrieval: Heat-induced epitope retrieval is typically recommended, though specific buffer conditions should be optimized.

• Detection System: Use a detection system appropriate for rabbit primary antibodies, such as polymer-based systems with HRP.

The Human Protein Atlas provides extensive immunohistochemical images for ATP6V1H across various tissues, which can serve as valuable reference data for expected staining patterns .

How can I validate the specificity of ATP6V1H antibodies in my experimental system?

Validating antibody specificity is critical for reliable research outcomes. For ATP6V1H antibodies, consider these validation approaches:

• Recombinant Expression Systems: Compare detection in ATP6V1H-transfected versus non-transfected cell lines, as demonstrated with H00051606-D01P antibody .

• Enhanced Validation Techniques: Some commercial antibodies like HPA023421 have undergone enhanced validation through recombinant expression systems .

• Immunogen Consideration: Verify that the immunogen used to generate the antibody matches your species of interest. For example:

  • ab187706 was generated against a synthetic peptide within human ATP6V1H aa 400-450

  • H00051606-D01P was raised against full-length human ATP6V1H protein (1-483 aa)

  • ab254685 targets a recombinant fragment within human ATP6V1H from aa 400 to C-terminus

• Cross-Reactivity Assessment: If working with non-human species, confirm cross-reactivity. For instance, ab187706 has been validated to work with both human and mouse samples .

• Knockdown/Knockout Controls: When possible, include ATP6V1H knockdown or knockout samples as additional specificity controls.

• Protein Array Testing: Some antibodies like HPA023421 have been tested against protein arrays containing 364 human recombinant protein fragments to assess cross-reactivity .

A multi-faceted validation approach will provide the highest confidence in antibody specificity for ATP6V1H detection.

How can I optimize immunoprecipitation protocols for ATP6V1H-protein interaction studies?

For ATP6V1H immunoprecipitation studies, consider these optimization strategies:

• Antibody Selection: Choose antibodies specifically validated for immunoprecipitation, such as ab187706, which has been confirmed suitable for IP applications with human and mouse samples .

• Complex Preservation: Since ATP6V1H functions within the V-ATPase complex, use lysis conditions that preserve protein-protein interactions:

  • Avoid harsh detergents that may disrupt the complex

  • Consider using buffers with mild detergents like 0.5% NP-40 or 1% Digitonin

  • Maintain physiological salt concentrations (around 150mM NaCl)

• Pre-clearing Strategy: To reduce non-specific binding, pre-clear lysates with protein A/G beads before adding the ATP6V1H antibody.

• Antibody Binding Conditions: Allow sufficient incubation time (4-16 hours at 4°C) for optimal antibody-antigen interactions.

• Elution Considerations: For downstream applications like mass spectrometry, consider native elution methods rather than denaturing conditions to maintain interacting partner integrity.

• Controls: Include critical controls:

  • IgG control from same species as the ATP6V1H antibody

  • Input sample (pre-IP lysate)

  • If possible, ATP6V1H-depleted samples as negative controls

When studying specific interactions between ATP6V1H and other V-ATPase components or novel interacting partners, optimizing these parameters will help ensure specific and biologically relevant results.

What approaches are recommended for studying ATP6V1H in the context of metabolic disorders like type 2 diabetes?

Recent research has implicated ATP6V1H in metabolic regulation, particularly in relation to type 2 diabetes. When investigating this connection, consider these specialized approaches:

• Expression Analysis in Disease Models:

  • ATP6V1H expression has been found to be down-regulated in human pancreatic islets associated with type 2 diabetes

  • Its expression is negatively associated with HbA1c levels, a key diabetes marker

• Functional Studies in Pancreatic Islets:

  • ATP6V1H has a positive regulatory effect on glucose-stimulated insulin secretion

  • Consider using appropriate pancreatic β-cell lines or primary islet cultures

• Antibody Selection for Pancreatic Tissue:

  • For human pancreatic tissues, HPA023421 has been extensively validated through the Human Protein Atlas project

  • Ensure antibodies are compatible with fixation methods typically used for pancreatic tissue

• Correlation Analysis:

  • Design experiments to correlate ATP6V1H expression levels with:

    • Insulin secretion capacity

    • HbA1c levels

    • Other diabetes biomarkers

• Mechanistic Investigations:

  • Study the relationship between V-ATPase function in endosomes/lysosomes and insulin secretion pathways

  • Investigate potential connections to autophagy, which is increasingly recognized in pancreatic cancer metabolism

• Comparative Studies:

  • Compare ATP6V1H expression and localization between diabetic and non-diabetic samples

  • Consider ethnic variations, as some studies have focused on specific populations like Mexican Americans

This research direction represents an emerging area connecting ATP6V1H function to metabolic disorders beyond its classical role in vesicular acidification.

How can I effectively troubleshoot background or non-specific staining with ATP6V1H antibodies?

When encountering background or non-specific staining with ATP6V1H antibodies, implement these troubleshooting strategies:

• Antibody Dilution Optimization:

  • For HPA023421 in IHC applications, try the recommended range of 1:50-1:200

  • For immunofluorescence with HPA023421, use 0.25-2 μg/mL

  • For Western blot with HPA023421, use 0.04-0.4 μg/mL

  • Always perform a dilution series to determine optimal concentration

• Blocking Protocol Enhancement:

  • Extend blocking time (1-2 hours at room temperature)

  • Try different blocking agents (5% BSA, 5% normal serum from the secondary antibody host species)

  • Consider adding 0.1-0.3% Triton X-100 for better penetration in IHC/IF applications

• Washing Stringency:

  • Increase number of wash steps

  • Extend washing times

  • Add 0.1% Tween-20 to wash buffers

• Antibody Specificity Verification:

  • Perform peptide competition assays using the immunizing peptide

  • Include ATP6V1H-depleted samples as negative controls

• Secondary Antibody Considerations:

  • Ensure secondary antibody is appropriate for the host species (rabbit for all listed ATP6V1H antibodies)

  • Try highly cross-adsorbed secondary antibodies to reduce cross-reactivity

  • Use secondary-only controls to identify background from the detection system

• Tissue-Specific Optimizations:

  • Adjust fixation protocols based on tissue type

  • Optimize antigen retrieval methods (citrate buffer vs. EDTA buffer, pH variations)

  • Consider tissue-specific autofluorescence quenching methods for IF applications

Following these systematic troubleshooting approaches will help achieve optimal signal-to-noise ratio for ATP6V1H detection across various applications.

How does ATP6V1H expression pattern vary across different tissues and cell types?

ATP6V1H expression shows distinct patterns across tissues and cell types, which is important to consider when interpreting experimental results:

• Tissue Distribution: While specific expression data isn't detailed in the provided search results, the Human Protein Atlas project (which validated the HPA023421 antibody) has tested ATP6V1H expression across 44 normal human tissues and 20 cancer tissue types . Researchers can access this comprehensive tissue expression data through the Human Protein Atlas portal.

• Cellular Localization: ATP6V1H functions as part of the V-ATPase complex, which:

  • Primarily localizes to intracellular compartments like endosomes and lysosomes

  • In some specialized cell types, may be targeted to the plasma membrane

• Expression in Metabolic Tissues:

  • ATP6V1H has been specifically studied in pancreatic islets, where its expression levels correlate with glucose-stimulated insulin secretion

  • Down-regulation of ATP6V1H in human pancreatic islets has been associated with type 2 diabetes

• Subcellular Distribution: Through the Human Cell Atlas project efforts, immunofluorescence characterization has mapped ATP6V1H at the subcellular level .

• Correlation with Function:

  • ATP6V1H expression is functionally associated with endosomal acidification

  • Its presence is essential for V-ATPase activity but not for complex assembly

  • In pancreatic tissues, expression correlates negatively with HbA1c levels

When interpreting ATP6V1H expression data, these tissue-specific and subcellular patterns should be considered alongside the biological question being investigated.

What experimental controls are essential when studying ATP6V1H antibody specificity?

To ensure reliable results when working with ATP6V1H antibodies, implement these essential controls:

• Positive Controls:

  • Cell lines or tissues known to express ATP6V1H at detectable levels

  • Recombinant ATP6V1H-expressing systems, as demonstrated with H00051606-D01P validation

  • For studies examining disease states, appropriate disease and normal tissue pairs

• Negative Controls:

  • For overexpression studies: non-transfected lysates, as shown in H00051606-D01P validation

  • When possible, ATP6V1H-knockdown or knockout samples

  • IgG controls from the same species as the primary antibody (rabbit for all ATP6V1H antibodies described)

• Specificity Controls:

  • Peptide competition assays using the immunizing peptide

  • For ab187706: peptide within human ATP6V1H aa 400-450

  • For H00051606-D01P: peptides from the full-length protein (aa 1-483)

  • For ab254685: peptides from aa 400 to C-terminus

• Technical Controls:

  • Secondary antibody-only controls to assess background staining

  • Loading controls for Western blot (housekeeping proteins)

  • Isotype controls for immunoprecipitation

  • Tissue processing controls for IHC/IF (known antigens with established patterns)

• Validation in Multiple Applications: When possible, confirm findings using multiple techniques (e.g., IF, WB, IHC) to increase confidence in results.

The Human Protein Atlas project provides extensive validation data for antibodies like HPA023421, including testing on protein arrays containing 364 human recombinant protein fragments to assess cross-reactivity .

How do different experimental approaches for ATP6V1H detection complement each other?

Different experimental techniques provide complementary information about ATP6V1H expression, localization, and function:

The Human Protein Atlas project exemplifies this complementary approach by systematically testing antibodies like HPA023421 across multiple applications, including IHC on tissue arrays and IF for subcellular mapping . For comprehensive ATP6V1H characterization, researchers should consider employing multiple detection methods, selecting antibodies specifically validated for each application.

What is the connection between ATP6V1H and type 2 diabetes pathophysiology?

Emerging research has revealed intriguing connections between ATP6V1H and type 2 diabetes:

• Expression Correlation: ATP6V1H gene expression is down-regulated in human pancreatic islets associated with type 2 diabetes .

• Glycemic Control Marker: ATP6V1H expression shows a negative correlation with HbA1c levels, a key marker of long-term glycemic control .

• Functional Impact: ATP6V1H has been demonstrated to have a positive regulatory effect on glucose-stimulated insulin secretion .

• Potential Mechanism: As part of the V-ATPase complex, ATP6V1H contributes to vesicular acidification, which is crucial for insulin granule maturation and exocytosis in pancreatic β-cells.

• Population-Specific Studies: Research has examined this relationship in specific populations, including Mexican Americans, suggesting potential ethnic variations in this association .

• Research Direction: These findings position ATP6V1H as a potential target for investigating mechanistic links between endosomal/lysosomal function and insulin secretion pathways.

The evidence suggesting ATP6V1H involvement in glucose homeostasis and insulin secretion adds a new dimension to understanding the diverse cellular processes implicated in type 2 diabetes pathophysiology.

How can ATP6V1H antibodies be utilized in cancer research applications?

ATP6V1H antibodies offer valuable tools for investigating cancer-related processes:

• Expression Profiling: Through the Human Protein Atlas project, antibodies like HPA023421 have been extensively validated on tissue arrays containing 20 of the most common cancer types , enabling systematic analysis of ATP6V1H expression across different cancer types.

• Acidic Microenvironment Studies: Since V-ATPases contribute to acidification of the tumor microenvironment, ATP6V1H antibodies can help investigate this aspect of cancer metabolism.

• Autophagy Research: Recent research has implicated autophagy-lysosome function in pancreatic cancer metabolism . ATP6V1H antibodies can help study the role of V-ATPase in this process.

• Cellular Stress Response: V-ATPase function relates to cellular stress response pathways that maintain metabolic homeostasis, which is emerging as a critical growth and survival mechanism in many cancers .

• Therapeutic Target Investigation: Changes in ATP6V1H expression or localization in response to anticancer therapies can be monitored using validated antibodies.

• Diagnostic Applications: Expression patterns detected using antibodies like HPA023421 could potentially contribute to cancer subtyping or prognostic assessments.

Given that pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy , the study of ATP6V1H and V-ATPase function in this context represents a particularly promising research direction where these antibodies can make significant contributions.

What emerging methodologies are being applied to study ATP6V1H in complex biological systems?

Several advanced methodologies are being employed to study ATP6V1H in complex biological contexts:

• Multi-omics Integration:

  • Combining ATP6V1H protein expression data (using antibodies) with transcriptomic analyses

  • Correlating protein levels with metabolomic profiles, particularly in diabetes research

• Advanced Imaging Techniques:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging to track dynamic V-ATPase complex assembly and function

  • The Human Cell Atlas project utilizes immunofluorescence to map ATP6V1H at the subcellular level

• Functional Genomics Approaches:

  • CRISPR-Cas9 genome editing to study ATP6V1H function

  • siRNA knockdown followed by antibody-based detection of pathway effects

• Protein-Protein Interaction Studies:

  • Proximity labeling techniques (BioID, APEX) coupled with ATP6V1H antibodies

  • Advanced co-immunoprecipitation approaches using antibodies like ab187706

• Tissue-Specific Conditional Models:

  • Investigating ATP6V1H function in specific tissues like pancreatic islets

  • Correlating expression with functional outcomes like insulin secretion

• Translational Applications:

  • Using ATP6V1H antibodies to investigate responses to therapeutic interventions

  • Exploring ATP6V1H as a potential biomarker in metabolic disorders

These emerging methodologies, combined with well-validated ATP6V1H antibodies, are expanding our understanding of this protein's roles in normal physiology and disease states, particularly in the contexts of cancer metabolism and diabetes pathophysiology .

What are the most important considerations when selecting an ATP6V1H antibody for specific research applications?

When selecting an ATP6V1H antibody for research, consider these critical factors:

• Application Compatibility: Match the antibody to your specific technique:

  • For Western blot: ab187706 , HPA023421 , H00051606-D01P , ab254685

  • For immunohistochemistry: HPA023421 , ab254685

  • For immunofluorescence: HPA023421 , ab254685

  • For immunoprecipitation: ab187706

• Species Reactivity: Ensure compatibility with your experimental system:

  • For human samples: All listed antibodies are validated

  • For mouse samples: ab187706 has demonstrated reactivity

  • For other species: Check cross-reactivity data or sequence homology

• Epitope Location: Consider the antibody's target region:

  • ab187706: Targets aa 400-450

  • H00051606-D01P: Raised against full-length protein (aa 1-483)

  • ab254685: Targets aa 400 to C-terminus

• Validation Extent: Assess the comprehensiveness of validation:

  • HPA023421: Extensively validated through the Human Protein Atlas project on tissue arrays and protein arrays

  • H00051606-D01P: Validated in transfected versus non-transfected systems

• Formulation Requirements: Consider experimental constraints:

  • Antibodies like H00051606-D01P are available in azide-free and BSA-free formulations

• Research Context: Match the antibody to your biological question:

  • For metabolism/diabetes studies: HPA023421 has been used in this context

  • For cancer research: HPA023421 has been tested across cancer tissue types

• Technical Support: Consider vendors that provide comprehensive protocols and troubleshooting guidance

By carefully evaluating these factors, researchers can select the most appropriate ATP6V1H antibody for their specific experimental needs, enhancing the reliability and significance of their findings.

How might ATP6V1H research evolve in the coming years?

ATP6V1H research is likely to evolve in several promising directions:

• Expanded Metabolic Disease Connections: Building on the association with type 2 diabetes , future research will likely explore ATP6V1H's role in other metabolic disorders and insulin resistance mechanisms.

• Cancer Metabolism Insights: The connection between autophagy-lysosome function and cancer metabolism will drive further investigation of ATP6V1H in various cancer types, potentially revealing new therapeutic approaches.

• Tissue-Specific Functions: More detailed characterization of ATP6V1H's tissue-specific roles, building on the comprehensive mapping from projects like the Human Protein Atlas .

• Structure-Function Relationships: Advanced structural biology techniques may reveal how ATP6V1H regulates V-ATPase activity at the molecular level.

• Therapeutic Targeting: Development of approaches to modulate V-ATPase function through ATP6V1H for potential therapeutic applications in diabetes or cancer.

• System-Level Integration: Investigation of ATP6V1H within broader cellular networks, connecting vesicular acidification to other cellular processes like autophagy, endocytosis, and secretion.

• Population Genetics: Exploration of genetic variations in ATP6V1H across different populations and their relationship to disease susceptibility, building on studies in specific populations like Mexican Americans .