ATP6V1B1 Antibody

What is ATP6V1B1 and what is its primary function in cellular physiology?

ATP6V1B1, also known as ATP6B1, VATB, and VPP3, is a component of vacuolar ATPase (V-ATPase), a multisubunit enzyme that mediates acidification of eukaryotic intracellular organelles. It belongs to the ATPase alpha/beta chains family and is primarily expressed in kidney tissues. ATP6V1B1 plays an essential role in the proper assembly and activity of V-ATPase . In renal intercalated cells, it mediates secretion of protons (H+) into the urine, ensuring correct urinary acidification . Additionally, the V-ATPase B1 isoform functions in proton secretion in the inner ear and is required to maintain proper endolymph pH and normal auditory function .

What is the molecular structure and characteristics of the ATP6V1B1 protein?

ATP6V1B1 is part of the cytosolic V1 domain of the V-ATPase complex, which consists of three A and three B subunits, two G subunits, plus the C, D, E, F, and H subunits. The V1 domain contains the ATP catalytic site . The calculated molecular weight of ATP6V1B1 is approximately 57 kDa, while the observed molecular weight is typically 56 kDa . The protein is encoded by the ATP6V1B1 gene located on chromosome 2cen-q13 in humans (Gene ID: 525), and its UniProt ID is P15313 .

What are the optimal applications and dilutions for ATP6V1B1 antibodies in different experimental techniques?

ATP6V1B1 antibodies can be used in multiple research applications with specific dilution recommendations:

It is generally recommended that these reagents should be titrated in each testing system to obtain optimal results, as detection sensitivity may be sample-dependent . For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, or alternatively, citrate buffer pH 6.0 may be used .

How should sample preparation be optimized for ATP6V1B1 antibody-based detection methods?

For optimal ATP6V1B1 detection in tissue samples via immunohistochemistry, tissue microarray (TMA) construction with tissue cores of 1.0 mm in diameter containing a sufficient proportion of tumor cells is recommended. Following FFPE tissue sectioning to 5-μm thickness, sections should be deparaffinized with xylene and rehydrated in serially graded ethanol to distilled water. For antigen retrieval, it is advised to incubate TMA sections in a steam pressure cooker containing heat-activated antigen retrieval buffer at pH 6.0 at 125°C for 2 min .

For Western blotting, positive detection has been reported in various tissue samples including mouse brain tissue, mouse kidney tissue, rat brain tissue, human brain tissue, pig brain tissue, pig cerebellum tissue, rabbit brain tissue, rabbit cerebellum tissue, and JAR cells . For immunofluorescence applications, HEK-293 cells have been verified as suitable samples .

How is ATP6V1B1 expression correlated with disease progression and prognosis in cancer research?

Research has demonstrated significant upregulation of ATP6V1B1 in epithelial ovarian cancer (EOC) compared with borderline and benign tumors and nonadjacent normal epithelial tissues. High ATP6V1B1 expression has been associated with several adverse clinicopathological parameters in EOC, including:

• Serous cell type

• Advanced International Federation of Gynecology and Obstetrics stage

• High/advanced tumor grade

• Elevated serum cancer antigen 125 levels

• Platinum resistance (P=0.011)

What methods can be employed to investigate ATP6V1B1's role in acid-base homeostasis and renal function?

To investigate ATP6V1B1's role in acid-base homeostasis, researchers can employ genetic models such as ATP6V1B1 knockout mice subjected to acid loading tests. In one study approach, Atp6v1b1+/+, Atp6v1b1+/-, and Atp6v1b1-/- mice were subjected to an HCl-load for 7 days to investigate acid-base status, kidney function, and expression of renal acid-base transport proteins .

Key parameters to measure include:

• Urinary pH and ammoniuria

• Blood chloride levels and pCO2

• Subcellular localization of other H+-ATPase subunits (a4 and B2)

• Expression levels of B1, B2, and a4 in renal membrane fractions

• Compensatory mechanisms such as regulation of pendrin in the collecting duct

Such studies have revealed that Atp6v1b1-/- mice exhibit more alkaline urine and low ammoniuria, whereas Atp6v1b1+/- mice show differences in blood parameters like higher blood chloride and lower blood pCO2, indicating a mild incomplete distal renal tubular acidosis (dRTA) that is partly compensated by respiration .

What are common challenges in ATP6V1B1 antibody detection and how can they be addressed?

Common challenges in ATP6V1B1 antibody detection include:

• Specificity issues: To ensure specificity, perform proper controls. For negative controls, use IgG in place of primary antibody to evaluate nonspecific staining. Include appropriate positive control specimens in your experimental design .

• Antigen accessibility: Proper antigen retrieval is crucial. For immunohistochemistry, using a steam pressure cooker with heat-activated antigen retrieval buffer at pH 6.0 or TE buffer at pH 9.0 is recommended. For difficult samples, experiment with different antigen retrieval methods and buffers .

• Signal intensity variation: Antibody dilution needs to be optimized for each experimental system. It is advisable to test a range of dilutions to determine the optimal concentration that provides specific signal with minimal background .

• Sample-dependent variation: Detection sensitivity can vary based on the sample type. Always validate the antibody with both positive and negative control samples relevant to your experimental design .

How can researchers validate the specificity of ATP6V1B1 antibodies in their experimental systems?

To validate ATP6V1B1 antibody specificity:

• Knockout/knockdown controls: Utilize ATP6V1B1 knockout or knockdown samples as negative controls. The CRISPR/Cas9 system has been used to generate ATP6V1B1-knockout cells that can serve as validation tools .

• Multiple antibody comparison: Use multiple antibodies targeting different epitopes of ATP6V1B1 to confirm staining patterns.

• Cross-reactivity testing: Verify antibody specificity on a protein array containing the target protein plus non-specific proteins. Some commercial antibodies have been verified against arrays containing 383 non-specific proteins .

• Multiple detection methods: Confirm results using different detection techniques (e.g., WB, IHC, IF) to ensure consistent identification of the target protein.

• Mass spectrometry verification: For definitive validation, immunoprecipitate the protein using the antibody and confirm its identity through mass spectrometry.

How can ATP6V1B1 antibodies be utilized to investigate intracellular pH regulation mechanisms?

ATP6V1B1 antibodies can be valuable tools for investigating intracellular pH regulation by:

• Localization studies: Using immunofluorescence techniques with ATP6V1B1 antibodies to track changes in V-ATPase distribution under various physiological and pathological conditions.

• Co-localization experiments: Combining ATP6V1B1 antibodies with pH-sensitive fluorescent probes to correlate V-ATPase distribution with local pH changes in subcellular compartments.

• Knockout/knockdown studies: Using ATP6V1B1 antibodies to confirm protein depletion in genetic models, followed by measurement of intracellular pH. For example, ATP6V1B1-knockout SKBR3 and JIMT-1 cells showed significantly lower intracellular pH compared to control cells .

• Protein complex formation: Employing ATP6V1B1 antibodies in co-immunoprecipitation studies to investigate how V-ATPase assembly and interaction with other proteins affect pH regulation.

• Therapeutic targeting assessment: Using these antibodies to evaluate the efficacy of pH-modulating interventions in disease models, particularly in cancer research where acidification plays a significant role in treatment resistance.

What experimental approaches can be used to study ATP6V1B1's role in cancer cell behaviors and therapeutic resistance?

To investigate ATP6V1B1's role in cancer progression and therapy resistance, researchers can employ these approaches:

• Expression analysis in patient samples: Use ATP6V1B1 antibodies for immunohistochemistry on tissue microarrays to correlate expression levels with clinical parameters and treatment outcomes. This approach has revealed that high ATP6V1B1 expression is associated with platinum resistance in EOC (P=0.011) .

• Genetic manipulation studies: Generate ATP6V1B1 knockout cancer cell lines using CRISPR/Cas9 technology to study phenotypic changes. Knockdown of ATP6V1B1 has been shown to decrease cancer cell proliferation and colony-forming abilities by inducing cell cycle arrest in G0/G1 phase .

• Intracellular pH and enzyme activity assessment: Combine ATP6V1B1 antibody-based detection with pH measurements to understand how altered expression affects intracellular acidification and enzyme activities. For example, ATP6V1B1-knockout cells exhibited decreased intracellular pH, which affected granzyme dynamics during antibody-dependent cellular cytotoxicity reactions .

• Drug sensitivity profiling: Compare chemotherapy response between wild-type and ATP6V1B1-modified cells to understand the mechanistic basis of resistance.

• In vivo tumor models: Use ATP6V1B1 antibodies to analyze protein expression in xenograft models treated with various therapeutic regimens to validate findings from cell culture systems.

How might ATP6V1B1 antibodies contribute to understanding the relationship between V-ATPase activity and immune system function?

ATP6V1B1 antibodies can provide valuable insights into the relationship between V-ATPase activity and immune function through:

• Granzyme bioactivity studies: Research has shown that downregulation of ATP6V1B1 expression suppresses granzyme bioactivity by decreasing intracellular pH. ATP6V1B1 knockout cells exhibited accumulated granzymes during antibody-dependent cellular cytotoxicity (ADCC) reactions, suggesting a role in immune evasion mechanisms .

• Antigen presentation analysis: V-ATPases are crucial for maintaining optimal pH in endosomal compartments where antigen processing occurs. ATP6V1B1 antibodies can help visualize and quantify V-ATPase distribution in antigen-presenting cells under various immunological challenges.

• Tumor microenvironment studies: Examining ATP6V1B1 expression in tumor-associated immune cells compared to cancer cells could reveal differential pH regulation strategies that contribute to immune suppression in the tumor microenvironment.

• Checkpoint inhibitor combination studies: Investigating whether ATP6V1B1 modulation can enhance the efficacy of immunotherapies through altered pH-dependent processes in both cancer and immune cells.

What novel methodological approaches could enhance the utility of ATP6V1B1 antibodies in biomarker development and personalized medicine?

Emerging methodological approaches to enhance ATP6V1B1 antibody utility in precision medicine include:

• Multiplexed immunofluorescence: Combining ATP6V1B1 antibodies with other markers in multiplexed staining protocols to develop comprehensive biomarker signatures that predict treatment response more accurately than single markers.

• Single-cell analysis: Applying ATP6V1B1 antibodies in single-cell proteomics approaches to understand heterogeneity in V-ATPase expression within tumors and its impact on treatment outcomes.

• Liquid biopsy integration: Developing protocols to detect ATP6V1B1 in circulating tumor cells or extracellular vesicles to monitor treatment response non-invasively.

• Biomarker panel development: Including ATP6V1B1 in gene panels evaluated by next-generation/high-throughput sequencing technology. As noted in research, "including ATP6V1B1 in the subset of gene panels would be more effective to facilitate a personalized therapy and increase the survival of patients with EOC as new avenues of EOC molecular characterization were opened using next-generation/high-throughput sequencing technology to predict platinum resistance or prognosis" .

• Therapeutic targeting strategies: Using ATP6V1B1 antibodies to evaluate the efficacy of V-ATPase inhibitors or pH-modulating drugs in patient-derived models to guide treatment selection.