ATP6V1G3 Antibody

What is ATP6V1G3 and what cellular functions does it perform?

ATP6V1G3 (also known as ATP6G3 or V-ATPase subunit G3) is a catalytic subunit of the peripheral V1 complex of vacuolar ATPase (V-ATPase). V-ATPase is responsible for acidifying various intracellular compartments in eukaryotic cells . The V-ATPase complex consists of two major domains: a cytosolic V1 domain that hydrolyzes ATP and a membrane-integral V0 domain that translocates protons .

The V1 domain consists of three A and three B subunits, two G subunits (one of which is ATP6V1G3), plus the C, D, E, F, and H subunits . This domain contains the ATP catalytic site. V-ATPase-dependent organelle acidification is necessary for critical intracellular processes including:

• Protein sorting

• Zymogen activation

• Receptor-mediated endocytosis

• Synaptic vesicle proton gradient generation

In some cell types, V-ATPase is targeted to the plasma membrane, where it is responsible for acidifying the extracellular environment .

What is the molecular weight of ATP6V1G3 protein and how is it typically detected?

As noted by Elabscience: "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. The common factors include: If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane."

What species reactivity do commercially available ATP6V1G3 antibodies typically have?

Most commercially available ATP6V1G3 antibodies show reactivity with human and mouse samples . Some antibodies may also react with rat samples . When selecting an antibody for your research, it's essential to verify the specific reactivity of each product, as this can vary between manufacturers and even between different clones from the same manufacturer.

Reactivity information is typically provided in product data sheets and should be confirmed experimentally before proceeding with critical experiments, especially when working with less common model organisms.

In which tissues and cell types is ATP6V1G3 most highly expressed?

ATP6V1G3 expression is relatively restricted compared to other V-ATPase subunits. According to the search results, ATP6V1G3 is primarily expressed in:

• Kidney tissue

• Inner ear

This restricted expression pattern makes ATP6V1G3 a potentially useful marker for certain tissue types and cellular structures. In kidney tissue, ATP6V1G3 plays a major role in differentiating between chromophobe renal cell carcinoma (RCC) and other subtypes of RCC . It has been described as "a highly delicate and particular immunohistochemical marker for chromophobe RCC and oncocytoma" .

Experimental Applications

For optimal results in immunohistochemistry applications with ATP6V1G3 antibodies, antigen retrieval is a critical step. Based on the search results, the following antigen retrieval methods have been recommended:

• Heat-mediated antigen retrieval with citrate buffer (pH 6.0)

• Alternative method: Heat-mediated antigen retrieval with TE buffer (pH 9.0)

The specific protocol from Proteintech recommends:
"For IHC suggested antigen retrieval with TE buffer pH 9.0; alternatively, antigen retrieval may be performed with citrate buffer pH 6.0"

For detailed protocols, researchers should refer to the manufacturer's specific recommendations for the antibody they are using, as optimal conditions may vary between different antibody preparations.

How should ATP6V1G3 antibodies be stored to maintain optimal activity?

Proper storage is essential for maintaining antibody activity and extending shelf life. For ATP6V1G3 antibodies, the following storage recommendations are commonly provided:

• Store at -20°C for long-term storage

• Valid for approximately 12 months when properly stored

• Avoid repeated freeze-thaw cycles to prevent activity loss

• For short-term storage (up to 2 weeks), refrigeration at 2-8°C is acceptable

Many manufacturers provide the antibody in a storage buffer containing glycerol (typically 50%) and a preservative such as sodium azide (0.02-0.09%) to help maintain stability . This formulation helps prevent freezing damage during storage at -20°C and extends the antibody's shelf life.

How can I validate the specificity of ATP6V1G3 antibody in my experimental system?

Validating antibody specificity is crucial for generating reliable research data. For ATP6V1G3 antibodies, consider the following validation approaches:

• Positive and negative control tissues: Use kidney tissue as a positive control since ATP6V1G3 is known to be expressed there . Tissues with minimal ATP6V1G3 expression can serve as negative controls.

• Knockdown/knockout validation: If possible, use samples from ATP6V1G3 knockdown or knockout systems to confirm that the signal disappears or is significantly reduced.

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody is capturing the intended protein.

• Orthogonal validation: Compare results using different antibodies targeting different epitopes of ATP6V1G3, or validate with orthogonal methods like RNA-seq as mentioned for the Sigma antibody product (HPA028701) .

• Western blot analysis: Check for a single band of appropriate molecular weight (~14 kDa) in tissues known to express ATP6V1G3 .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to verify that this blocks specific binding.

Some manufacturers like Boster Bio mention that they validate all antibodies with "known positive control and negative samples to ensure specificity and high affinity" .

What role does ATP6V1G3 play in recurrent spontaneous abortion, and how can antibodies be used to study this connection?

Recent research has identified ATP6V1G3 as a potential key gene in recurrent spontaneous abortion (RSA). According to a 2020 study analyzing gene expression data from GSE22490 and GSE26787 datasets:

• ATP6V1G3 was identified as a hub gene among the 46 differentially expressed genes (DEGs) between patients with recurrent miscarriage and patients with uncomplicated pregnancies .

• Protein expression analysis showed higher rates of ATP6V1G3 in both placental villus and decidual tissue in RSA cases compared to controls:

  Tissue type	Recurrent group (n=31)	Control group (n=30)
  Placental villus - High expression	13 (43.3%)	7 (23.3%)
  Placental villus - Low expression	18	23
  Decidual tissue - High expression	10 (33.3%)	5 (16.7%)
  Decidual tissue - Low expression	21	25

• Although the differences were not statistically significant (p>0.05), potentially due to small sample size, the consistently higher expression in RSA cases suggests ATP6V1G3 may be involved in RSA pathogenesis .

• Gene ontology analysis of the differentially expressed genes indicated enrichment in biological processes including glutamate secretion, positive regulation of synapse assembly, T cell receptor signaling pathway, and immune response .

• KEGG pathway analysis showed the dysregulated genes were enriched in the glutamatergic synapse pathway .

Researchers interested in studying this connection can use ATP6V1G3 antibodies for:

• Immunohistochemical analysis of placental and decidual tissues

• Western blot analysis to quantify expression levels

• Developing potential diagnostic markers for RSA risk

What are the optimal fixation protocols for detecting ATP6V1G3 in tissue samples?

For optimal detection of ATP6V1G3 in tissue samples, proper fixation is essential. Based on the available information, the following fixation protocol has been used successfully:

• Paraffin embedding protocol:

  • Fix tissue specimens in an appropriate fixative (typically 10% neutral buffered formalin)

  • Process and embed in paraffin following standard protocols

  • Cut sections to 4-7 μm thickness

• Deparaffinization and rehydration:

  • Soak slides in xylene for 10 min

  • Rehydrate through graded alcohols: absolute ethanol for 5 min, followed by 95%, 80%, and 79% ethanol for 5 min each

• Peroxidase blocking and antigen retrieval:

  • Incubate specimens in 3% H₂O₂ in the dark for 15 min to inactivate endogenous peroxidase

  • Perform antigen retrieval using 0.01 mol/L sodium citrate at 95°C for 15 min

  • Alternative: Use TE buffer (pH 9.0) as suggested by some manufacturers

• Blocking and primary antibody incubation:

  • Block with normal serum (10%) for antigen sealing

  • Dilute primary antibody with 10% serum

  • Apply to tissue and incubate overnight at 4°C

• Wash, secondary antibody, and detection:

  • Wash with PBS

  • Apply appropriate secondary antibody

  • Develop with DAB staining

  • Counterstain, dehydrate, clear, and mount as per standard protocols

This protocol has been successfully applied in research examining ATP6V1G3 expression in placental villus and decidual tissue samples .

How can I troubleshoot weak or non-specific signals when using ATP6V1G3 antibody in Western blot applications?

When encountering issues with ATP6V1G3 detection in Western blot applications, consider the following troubleshooting approaches:

• For weak signals:

  • Increase antibody concentration: Try using a higher concentration of primary antibody (e.g., 1:500 instead of 1:1000)

  • Extend incubation time: Overnight incubation at 4°C may improve signal strength

  • Increase protein loading: Load more protein (30-50 μg) to enhance detection of low-abundance proteins

  • Use more sensitive detection systems: Try enhanced chemiluminescence (ECL) substrates designed for higher sensitivity

  • Optimize transfer conditions: Ensure efficient protein transfer to the membrane, especially for lower molecular weight proteins like ATP6V1G3 (~14 kDa)

• For non-specific signals:

  • Increase blocking time/concentration: Use 5% non-fat dry milk or BSA for 1-2 hours at room temperature

  • Optimize antibody dilution: Test a range of dilutions to find the optimal concentration that provides specific signal with minimal background

  • Increase washing steps: More thorough washing between antibody incubations can reduce background

  • Use more stringent wash buffers: Adding 0.1-0.3% Tween-20 to PBS can help reduce non-specific binding

  • Verify sample integrity: Ensure samples are fresh and properly prepared with protease inhibitors

• Special considerations for ATP6V1G3:

  • Use appropriate positive controls: HEK-293 cells and mouse kidney tissue have been validated as positive controls for ATP6V1G3 detection

  • Molecular weight verification: Ensure you're examining the correct region of the membrane for the 14 kDa band

  • Consider using a freshly prepared antibody aliquot to avoid potential issues from repeated freeze-thaw cycles

What are the best secondary antibodies to use with rabbit polyclonal ATP6V1G3 antibodies?

Most commercially available ATP6V1G3 antibodies are rabbit polyclonals . For optimal detection, compatible secondary antibodies should be selected based on the specific application and detection system. Based on the search results, the following secondary antibodies are recommended:

For Western Blot applications:

• Goat Anti-Rabbit IgG H&L Antibody (HRP) for chemiluminescent detection

• Goat Anti-Rabbit IgG H&L Antibody (AP) for alkaline phosphatase-based detection systems

For Immunohistochemistry applications:

• Appropriate HRP-conjugated secondary antibodies optimized for IHC detection systems

• DAB (3,3'-diaminobenzidine) is commonly used as the chromogen for visualization

For Immunofluorescence applications:

• Goat Anti-Rabbit IgG H&L Antibody (FITC) for fluorescent imaging

For other applications:

• Goat Anti-Rabbit IgG H&L Antibody (Biotin) for streptavidin-based detection systems

When selecting a secondary antibody, consider factors such as sensitivity requirements, detection method, potential cross-reactivity issues, and compatibility with your imaging or visualization system.

How does ATP6V1G3 protein expression vary across different physiological and pathological conditions?

Understanding the expression patterns of ATP6V1G3 across different conditions can provide insights into its biological roles and potential as a biomarker. Based on the available research:

• Tissue-specific expression:

  • ATP6V1G3 expression is relatively restricted, with highest expression in kidney and inner ear tissues

  • This restricted expression pattern makes it potentially valuable as a tissue-specific marker

• Pathological conditions:

  • Renal cell carcinoma: ATP6V1G3 serves as "a highly delicate and particular immunohistochemical marker for chromophobe RCC and oncocytoma"

  • Recurrent spontaneous abortion: Higher rates of ATP6V1G3 protein expression were observed in both placental villus (43.3% vs 23.3%) and decidual tissue (33.3% vs 16.7%) in RSA cases compared to controls, although the differences were not statistically significant (p>0.05)

• Potential mechanisms in disease:

  • In recurrent spontaneous abortion, the dysregulated genes including ATP6V1G3 were enriched in biological functions related to:

    • Glutamate secretion

    • Positive regulation of synapse assembly

    • T cell receptor signaling pathway

    • Ion transmembrane transport

    • Chemical synaptic transmission

    • Immune response

  • The only enriched KEGG pathway was the glutamatergic synapse pathway

• Research implications:

  • The upregulation of ATP6V1G3 in recurrent spontaneous abortion suggests it may play an important role in this condition's development

  • Further biological experiments are needed to explore the specific molecular function of ATP6V1G3 in recurrent spontaneous abortion

  • The potential connection to immune response and signaling pathways suggests ATP6V1G3 may have broader implications in immune-mediated conditions

What novel research applications are emerging for ATP6V1G3 antibodies?

Based on recent research findings and technological advancements, several emerging applications for ATP6V1G3 antibodies show promise:

• Biomarker development for recurrent spontaneous abortion:

  • The identification of ATP6V1G3 as a potential key gene in RSA suggests it could be developed as a diagnostic or prognostic biomarker

  • Future research may focus on developing standardized immunoassays to quantify ATP6V1G3 expression in clinical samples

• Renal cancer diagnostics:

  • ATP6V1G3's role as a marker for chromophobe renal cell carcinoma and oncocytoma could be further developed for clinical pathology applications

  • Multiplex immunohistochemistry panels incorporating ATP6V1G3 may improve diagnostic accuracy

• Targeting acidification mechanisms in disease:

  • As a component of V-ATPase involved in cellular acidification processes, ATP6V1G3 could be investigated in contexts where pH regulation is dysregulated

  • This could include cancer research, where tumor microenvironment acidification is a known factor in progression

• Study of glutamatergic signaling pathways:

  • The enrichment of ATP6V1G3 and related genes in the glutamatergic synapse pathway suggests potential applications in neuroscience research

  • This could lead to investigations of ATP6V1G3 in neurological conditions involving glutamate dysregulation

• Single-cell analysis applications:

  • With advances in single-cell proteomics, ATP6V1G3 antibodies could be employed to study cellular heterogeneity in tissues where it is expressed

  • This may reveal previously unknown functions in specific cell populations

What are the key considerations when designing experiments to study ATP6V1G3 function in different cellular contexts?

When designing experiments to study ATP6V1G3 function, researchers should consider:

• Appropriate model selection:

  • Use tissues or cell lines known to express ATP6V1G3, such as kidney-derived cell lines or tissue samples

  • Consider the restricted expression pattern of ATP6V1G3 when selecting experimental models

  • Validated positive controls include HEK-293 cells and mouse kidney tissue

• Functional assays:

  • Design assays that assess V-ATPase activity and intracellular pH regulation

  • Consider the cellular compartments where ATP6V1G3 is likely to function

  • Measure effects on glutamate signaling, given the enrichment in glutamatergic synapse pathway

• Genetic manipulation approaches:

  • Use RNA interference (siRNA or shRNA) to knockdown ATP6V1G3 expression

  • Consider CRISPR-Cas9 gene editing for complete knockout studies

  • Rescue experiments with wild-type and mutant constructs can establish specificity

• Interaction studies:

  • Investigate protein-protein interactions with other V-ATPase components

  • The protein-protein interaction (PPI) network established in recurrent spontaneous abortion research identified 83 nodes and 273 edges with an average node degree of 6.58

  • Focus on interactions with other hub genes identified in relevant studies (ATP6V1E1, ATP6V0D2, ATP6V0B, and ATP6V1B1)

• Translational considerations:

  • For recurrent spontaneous abortion research, consider using both placental villus and decidual tissue samples

  • For renal cancer applications, carefully select appropriate positive and negative control tissues

  • Design experiments that can bridge basic research findings with potential clinical applications

By carefully considering these factors, researchers can design robust experiments that yield meaningful insights into ATP6V1G3 function in various physiological and pathological contexts.