ANXA6 Antibody

What are the validated applications for ANXA6 antibodies in current research?

ANXA6 antibodies have been validated for multiple research applications with varying specifications based on the particular antibody clone or product. Most commonly validated applications include:

When selecting an ANXA6 antibody for your experimental design, consider both the application compatibility and the specific epitope targeted. For instance, antibody CAB18069 is raised against a recombinant fusion protein containing amino acids 1-250 of human Annexin A6, while others may target different regions of the protein, potentially affecting specificity and cross-reactivity .

What is the expected molecular weight for ANXA6 detection in Western blot applications?

The expected molecular weight detection for ANXA6 in Western blot applications shows some variability in the literature:

• Theoretical/calculated molecular weight: 75,873 Da (≈76 kDa)

• Commonly observed band size: 76 kDa in most tissues

• Alternative observed band: 45 kDa reported in some samples

This discrepancy requires methodological consideration. The 76 kDa band represents the full-length protein, while the 45 kDa band might represent an alternatively spliced variant, proteolytic fragment, or post-translationally modified form. When performing Western blot analysis, researchers should validate their results using appropriate positive controls (such as HeLa cell lysate) and consider running reducing and non-reducing conditions to identify potential structural variations .

What is the proper storage and handling protocol for ANXA6 antibodies?

For optimal antibody performance and longevity, the following storage and handling protocols are recommended based on product specifications:

• Long-term storage: -20°C for up to one year from receipt date

• After reconstitution: 4°C for up to one month

• For extended use: Aliquot and store at -20°C for up to six months

• Critical note: Avoid repeated freeze-thaw cycles as this significantly reduces antibody activity

When working with lyophilized antibody formulations, proper reconstitution is essential. For example, adding 0.2 ml of distilled water to lyophilized PA1436 yields a concentration of 500 μg/ml . Proper reconstitution and aliquoting immediately upon receipt significantly extends antibody shelf-life and maintains consistent experimental results across studies.

How should researchers design validation experiments for ANXA6 antibodies?

A comprehensive validation strategy for ANXA6 antibodies should include multiple complementary approaches:

• Knockout/knockdown validation: Use ANXA6 knockout or knockdown samples as negative controls. Several antibodies like CAB18069 are specifically labeled as "KO Validated," indicating they've been tested against genetic knockout models to confirm specificity .

• Multi-application testing: Validate the antibody using at least two different techniques (e.g., Western blot and IHC) to confirm consistent target recognition.

• Cross-species reactivity assessment: Verify species reactivity claims by testing on samples from multiple organisms. For example, PA1436 is reported to react with human, mouse, and rat samples , while CAB18069 reacts with human and mouse samples .

• Epitope mapping considerations: Consider the immunogen sequence when interpreting results. For CAB18069, the immunogen corresponds to amino acids 1-250 of human ANXA6 (NP_001146.2), which may impact detection of specific isoforms or variants .

• Immunoprecipitation testing: For interaction studies, validate antibodies using immunoprecipitation followed by Western blot detection, as demonstrated in studies examining ANXA6 interaction with SNAP23 .

What controls should be incorporated when using ANXA6 antibodies for immunohistochemistry?

For rigorous immunohistochemical analysis with ANXA6 antibodies, the following controls should be implemented:

When evaluating IHC results, it's important to note that ANXA6 typically shows cytoplasmic localization but may also exhibit membrane immunoreactivity in certain cancer tissues . In pancreatic ductal adenocarcinoma, high ANXA6 IHC scores correlate with tumor budding at invasive fronts (p=0.082) and perineural invasion (p<0.0001) .

What methodological approaches are required for studying ANXA6 translocation to cellular membranes?

Investigating ANXA6 translocation requires specialized methodologies that account for its calcium-dependent membrane binding properties:

• Calcium-dependent experimental design: Include conditions with and without calcium treatment, as extracellular Ca²⁺ treatment induces ANXA6 translocation to various membrane structures .

• Subcellular fractionation: Perform careful fractionation to isolate plasma membrane, mitochondria, vesicles, and endosomal compartments where ANXA6 may localize.

• Immunogold transmission electron microscopy: Utilize this high-resolution approach to visualize precise subcellular localization of ANXA6 in different membrane structures, as demonstrated in studies with TNBC cells .

• Proximity ligation assays (PLA): Implement PLA to study interactions between ANXA6 and potential binding partners like SNAP23, which may mediate its membrane-associated functions .

• Live-cell imaging with fluorescently tagged ANXA6: Monitor dynamic translocation in response to calcium flux or other stimuli in real-time.

Research has demonstrated that ANXA6 can translocate to the plasma membrane, mitochondria, vesicles, and endosomes upon calcium stimulation, with implications for cellular signaling, membrane trafficking, and vesicle secretion processes .

How does ANXA6 expression modulate cancer cell invasion and metastasis?

ANXA6's role in cancer invasion and metastasis exhibits context-dependent complexity:

In breast cancer models, ANXA6 demonstrates complex regulatory effects:

• Silencing ANXA6 in invasive BT-549 breast cancer cells enhances anchor cell growth but strongly inhibits intercellular cohesion, cell adhesion/diffusion, cell motility, and invasiveness .

• Mechanistically, ANXA6 depletion strongly inhibits focal adhesion kinase and PI3K/AKT pathways while the MAPK pathway remains constitutively active .

• In mouse TNBC xenograft models, loss of ANXA6 is associated with tumorigenesis and development, suggesting ANXA6 may inhibit tumor proliferation in TNBC cells .

Function-blocking approaches provide additional insights:

• Monoclonal antibody 9E1 targeting ANXA6 significantly decreases invasion in pancreatic, lung squamous, and breast cancer cells .

• Silencing of ANXA6 leads to markedly reduced invasive capacity of pancreatic and lung squamous cancer in vitro .

Clinical correlations further support ANXA6's role in aggressive cancer:

• In pancreatic ductal adenocarcinoma, high ANXA6 IHC scores correlate with tumor budding at invasive fronts (p=0.082), perineural invasion (p<0.0001), and show a weak correlation with reduced survival (p=0.2242) .

These findings suggest ANXA6 may serve as both a biomarker for aggressive cancer behavior and a potential therapeutic target in specific cancer contexts.

What is the role of ANXA6 in regulating pro-inflammatory cytokine secretion?

Recent research has established ANXA6 as a critical regulator of inflammatory signaling in cancer cells:

ANXA6 regulates specific cytokine profiles:

• Reduced expression of ANXA6 inhibits the secretion of multiple cytokines, including DKK-1, IL-8, MCP-1, OPN, and TSP-1 in TNBC cells .

• Secretion of MCP-1 is consistently ANXA6-dependent in both BT-549 and MDA-468 TNBC cell lines .

The molecular mechanism involves secretory pathway components:

• ANXA6 interacts with SNAP23, a component of the SNARE complex, as demonstrated by co-immunoprecipitation, proximity ligation assays, and GST pull-down experiments .

• This interaction is essential for the secretion of both pro-inflammatory cytokines and extracellular vesicles (EVs) in TNBC cells .

Functional consequences extend to extracellular vesicle characteristics:

• ANXA6 expression status influences not only the secretion of EVs but also their cholesterol content .

• The translocation of ANXA6 to various membranes supports its role in vesicle trafficking and secretory processes .

These findings suggest targeted inhibition of ANXA6 could potentially modulate inflammatory signaling in cancer microenvironments.

How can researchers effectively use neutralizing antibodies against ANXA6 for functional studies?

Neutralizing antibodies against ANXA6 represent powerful tools for investigating its extracellular functions:

Implementation considerations:

• Mouse monoclonal anti-Annexin VI antibody (IgG2b κ, G-10) has been validated as an effective neutralizing antibody for ANXA6 .

• This antibody recognizes ANXA6 on the cell surface, as demonstrated in BT-549 cells that express relatively high levels of ANXA6 .

Dose-dependent and cell-specific effects:

• Anti-ANXA6 neutralizing antibodies dose-dependently reduce the viability of TNBC cells, with differential effects based on ANXA6 expression levels .

• Mesenchymal-like HCC70 and BT-549 cells (high ANXA6 expressors) showed greater resistance than epithelial MDA-468 cells (lower ANXA6 expressors) .

• Viability of BT-549 cells was slightly decreased at 10 μg/ml, while that of MDA-468 was significantly reduced at concentrations as low as 2 μg/ml .

Experimental controls:

• Always include isotype control antibodies matched to the neutralizing antibody (e.g., mouse IgG2b κ isotype for the G-10 antibody) .

• Include both high and low ANXA6-expressing cell lines to demonstrate specificity of effects.

These findings suggest extracellular ANXA6 plays an important role in cancer cell survival, with greater dependence in cells expressing lower levels of the protein.

How should researchers interpret conflicting data regarding ANXA6's role in different cancer types?

The seemingly contradictory roles of ANXA6 reported across various cancer studies require careful analytical consideration:

Contextual factors that may explain discrepancies:

• Cell-type specificity: ANXA6 functions differently in mesenchymal-like versus epithelial cancer cells .

• Expression level dependency: Effects of ANXA6 inhibition vary based on baseline expression levels, with low-ANXA6 cells showing greater sensitivity to neutralizing antibodies .

• Subcellular localization differences: ANXA6 exhibits both cytoplasmic and membrane localization, with different functional implications .

• Cancer stage dependency: ANXA6 may have different roles during initiation versus progression/invasion phases of cancer.

Analytical approaches to reconcile contradictions:

• Perform parallel knockdown and overexpression studies in the same cell lines to establish dose-dependent relationships.

• Integrate multiple experimental techniques (knockdown, neutralizing antibodies, overexpression) to comprehensively characterize ANXA6 function.

• Analyze subcellular localization of ANXA6 in all experimental contexts.

• Consider cancer subtype classifications beyond traditional histological categories.

The emerging model suggests ANXA6 may function as both a tumor suppressor in certain contexts and a promoter of invasion/metastasis in others, highlighting the importance of context-specific analysis in cancer biology.

What are the best practices for interpreting ANXA6 immunohistochemistry results in clinical samples?

When analyzing ANXA6 immunohistochemistry in patient samples, researchers should follow these evidence-based interpretive guidelines:

Scoring methodology considerations:

• Implement a standardized scoring system that accounts for both staining intensity and percentage of positive cells.

• Separately evaluate membrane and cytoplasmic ANXA6 localization, as membrane immunoreactivity may correlate with aggressive tumor behavior .

• Pay particular attention to heterogeneity within tumors, especially at invasive fronts where ANXA6 expression may have prognostic significance .

Clinicopathological correlation:

Tissue-specific considerations:

• Normal tissues generally show restricted ANXA6 expression .

• Aggressive tumor types exhibit high prevalence of membrane immunoreactivity .

• Consider physiological context (e.g., hypoxic regions) when interpreting ANXA6 expression patterns .

Technical validation:

• Always include known positive controls (e.g., human liver, placenta) and negative controls.

• When using new antibody lots, validate with Western blot of tissue lysates before proceeding with IHC interpretation.

By following these guidelines, researchers can generate more reliable and clinically relevant data from ANXA6 immunohistochemistry studies.

How can researchers investigate the relationship between ANXA6 and hypoxia in tumor microenvironments?

Hypoxia-ANXA6 interactions represent an emerging research area with important implications for cancer biology:

Experimental design considerations:

• Utilize hypoxia chambers (like Bactrox) with controlled oxygen levels to induce reproducible hypoxic conditions .

• Employ luciferase reporter assays with the ANXA6 promoter to directly assess transcriptional regulation under hypoxic conditions .

• Consider the timing of hypoxia exposure, as both acute and chronic hypoxia may differently affect ANXA6 expression.

Key parameters to measure:

• ANXA6 mRNA and protein expression levels under normoxic vs. hypoxic conditions

• ANXA6 subcellular localization changes in response to hypoxia

• Interaction partners of ANXA6 specific to hypoxic conditions

• Functional consequences including:

  • Cell viability and apoptosis resistance

  • Glucose uptake and metabolism

  • ATP production

  • ROS generation

Therapeutic implications:

• Investigate whether hypoxia-induced ANXA6 expression affects sensitivity to anti-cancer therapeutics like Lapatinib .

• Explore combinatorial approaches targeting both hypoxia signaling and ANXA6 function.

This research direction could provide valuable insights into how tumor microenvironmental conditions modulate ANXA6 functions and potentially identify new therapeutic vulnerabilities in hypoxic tumors.

What methodological approaches should be used to investigate ANXA6's role in extracellular vesicle biogenesis and cargo selection?

Investigating ANXA6's functions in extracellular vesicle (EV) biology requires specialized experimental approaches:

EV isolation and characterization techniques:

• Implement differential ultracentrifugation, size exclusion chromatography, or commercial isolation kits optimized for different EV populations.

• Characterize isolated EVs using:

  • Nanoparticle tracking analysis for size distribution and concentration

  • Transmission electron microscopy for morphology

  • Western blotting for EV markers (CD63, CD9, TSG101)

  • Proteomic analysis for comprehensive cargo profiling

ANXA6-specific investigative approaches:

• Generate ANXA6 knockdown and overexpression models to assess effects on EV quantity and quality .

• Employ proximity-based labeling techniques (BioID, APEX) to identify ANXA6-proximal proteins in EV biogenesis compartments.

• Utilize live-cell imaging with fluorescently tagged ANXA6 to visualize its involvement in EV formation.

• Examine ANXA6's interaction with SNAP23 and other SNARE proteins in the context of EV secretion .

Functional assessment of ANXA6-dependent EVs:

• Compare cholesterol content of EVs from control versus ANXA6-modulated cells .

• Analyze the inflammatory potential of these EVs using recipient cell assays.

• Investigate the transfer of ANXA6 itself via EVs between different cell populations.

Recent research demonstrates that ANXA6 not only influences EV secretion rates but also affects their cholesterol content and potentially their functional properties in recipient cells , suggesting an important role in intercellular communication within the tumor microenvironment.