ANXA7 Antibody

What is Annexin A7 and why is it important in cellular research?

Annexin A7 (ANXA7) is a calcium-dependent membrane-binding protein that plays crucial roles in various cellular processes, including membrane organization, vesicle trafficking, and exocytosis. It is particularly important in research due to its involvement in surfactant secretion in alveolar type II cells and its potential roles in tumor biology. ANXA7 has been detected in multiple cell types across human, mouse, and rat models, making it valuable for comparative studies in different experimental systems. Recent research has also implicated ANXA7 in regulation of cell proliferation, migration, and invasion, suggesting its potential significance in cancer research .

How can I confirm the specificity of my ANXA7 antibody?

The specificity of an ANXA7 antibody can be confirmed through multiple methods. Western blot analysis should reveal a single band at approximately 47-51 kDa, which is the expected molecular weight for ANXA7 . The specificity can be further verified through pre-incubation of the antibody with recombinant ANXA7 protein, which should result in loss of reactivity in subsequent assays . Additionally, using ANXA7 knockout or knockdown samples as negative controls in your experiments provides robust validation. Researchers have demonstrated antibody specificity by showing that purified antibodies to recombinant ANXA7 recognize a single band at approximately 47 kDa in lung tissue, cultured type II cells, and isolated lung lamellar bodies .

What are the typical molecular weights observed for ANXA7 in different species?

ANXA7 is typically detected at approximately 47-51 kDa across different species and cell types. Western blot analyses have shown that antibodies against ANXA7 detect bands at approximately 50 kDa in human cell lines (such as Jurkat and HeLa) . In mouse and rat samples, ANXA7 is typically observed at around 47 kDa . The slight variations in molecular weight may be attributed to different post-translational modifications or isoforms of the protein across species and tissues. For instance, the Proteintech ANXA7 antibody (10154-2-AP) reports observed molecular weights of both 47 kDa and 51 kDa .

What cell lines and tissues are most commonly used to study ANXA7?

Based on the research literature, the following cell lines and tissues are frequently used in ANXA7 studies:

These samples have been validated for ANXA7 detection using various techniques including Western blot, immunohistochemistry, and immunofluorescence .

What are the optimal conditions for Western blot detection of ANXA7?

For optimal Western blot detection of ANXA7, the following conditions are recommended:

• Sample preparation: Use reducing conditions for cell or tissue lysates

• Protein separation: SDS-PAGE with proteins that can resolve the 47-51 kDa range effectively

• Transfer: PVDF membrane is commonly used for ANXA7 detection

• Antibody dilution: 1:2000-1:16000 dilution is recommended for polyclonal antibodies like Proteintech 10154-2-AP

• Secondary antibody: For goat primary antibodies, use HRP-conjugated Anti-Goat IgG (e.g., R&D Systems HAF017); for rabbit primaries, use appropriate anti-rabbit HRP conjugates

• Buffer system: Immunoblot Buffer Group 2 has been validated for ANXA7 detection

Western blot analysis has successfully detected ANXA7 in multiple cell lines including Jurkat, HeLa, C2C12, and L6, with specific bands appearing at approximately 50 kDa under these conditions .

How can I perform co-localization studies with ANXA7 and other markers?

For co-localization studies with ANXA7 and other markers, the following methodological approach is recommended:

• Cell preparation: Fix cells using 4% paraformaldehyde for 10 minutes

• Blocking and permeabilization: Perform simultaneously for 30 minutes

• Sequential staining: When using antibodies from the same species (e.g., rabbit anti-ANXA7 and rabbit anti-SNAP23), employ a sequential staining protocol to avoid cross-reactivity

• For double staining with ANXA7:

  • First, stain for the first marker (e.g., SNAP23) using standard protocols

  • Then, block with biotin blocking agents for 30 minutes

  • Incubate overnight with biotinylated anti-ANXA7 antibodies (1:500 dilution)

  • Detect using streptavidin-AlexaFluor568 conjugated antibodies (1:500 dilution)

• Counterstain nuclei with DAPI (1 minute)

• Mount using anti-fade reagent

• Analyze using confocal laser scanning microscopy

This protocol has been validated for co-localization studies of ANXA7 with SNAP23 and ABCA3 in alveolar type II cells, demonstrating increased co-localization coefficients after stimulation with secretagogues like PMA and calcium ionophore A23187 .

What considerations are important for immunohistochemical detection of ANXA7?

For successful immunohistochemical (IHC) detection of ANXA7, researchers should consider the following:

• Antigen retrieval method: Two options have proven effective:

  • TE buffer at pH 9.0 (recommended)

  • Citrate buffer at pH 6.0 (alternative)

• Antibody dilution: 1:200-1:800 is typically optimal for IHC applications

• Tissue validation: The antibody has been validated in multiple tissues including:

  • Mouse heart, stomach, and brain tissues

  • Human pancreas, prostate, and stomach tissues

  • Rat stomach and brain tissues

• Controls: Include positive control tissues known to express ANXA7 and negative controls (omitting primary antibody)

• Detection system: Use an appropriate secondary antibody system compatible with the primary antibody host species

• Counterstaining: Hematoxylin is commonly used for nuclear counterstaining in ANXA7 IHC

Researchers should optimize these conditions for their specific tissue of interest, as expression levels and accessibility of ANXA7 epitopes may vary across different tissues and fixation methods .

How can ANXA7 knockdown be achieved for functional studies?

To achieve effective ANXA7 knockdown for functional studies, the following methodological approach can be implemented:

• Design of shRNA sequences:

  • Search for the ANXA7 gene sequence in gene banks (reference sequence: NM_009674.3 for mouse)

  • Analyze mRNA spatial accessibility and free energy properties

  • Exclude sequences with potential off-target effects

  • Design appropriate shRNA and negative control (unrelated) sequences

• Vector preparation:

  • Use a suitable vector system (e.g., pGPU6/GFP/Neo)

  • Perform linearization of the vector

  • Construct the expression vector by ligating the shRNA template

• Transfection:

  • Optimize transfection conditions for your specific cell line

  • Confirm knockdown efficiency using Western blot and/or qPCR

• Validation controls:

  • Include non-transfected cells as wild-type controls

  • Include cells transfected with non-targeting shRNA sequences

This approach has been successfully used to down-regulate ANXA7 in Hca-P cells, resulting in decreased expression of ANXA7 and its related protein SODD, which subsequently reduced tumor cell migration, invasion, and proliferation .

What functional assays can be used to study the effects of ANXA7 modulation?

Several functional assays can be employed to investigate the effects of ANXA7 modulation:

• Migration assays:

  • Transwell cell transfer experiments to measure cell migration

  • Wound healing assays to assess collective cell migration

• Invasion assays:

  • Matrigel-coated Transwell chambers to evaluate invasive potential

  • 3D spheroid invasion assays for more physiologically relevant models

• Proliferation assays:

  • CCK8 assay measured at multiple time points (e.g., 0, 24, and 48 hours)

  • BrdU incorporation assay to measure DNA synthesis

• Exocytosis/secretion assays:

  • Measure surfactant secretion in alveolar type II cells

  • Monitor membrane trafficking using fluorescent markers

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify ANXA7 binding partners

  • Proximity ligation assays to visualize protein interactions in situ

Research has demonstrated that down-regulation of ANXA7 significantly reduces tumor cell penetration through cell membranes and Matrigel, and slows proliferation rates as measured by CCK8 assay . Additionally, stimulation with agents like PMA or calcium ionophore A23187 has been shown to increase ANXA7's membrane association and co-localization with proteins like SNAP23, suggesting its role in membrane trafficking and exocytosis .

How does ANXA7 trafficking change in response to cellular stimulation?

ANXA7 trafficking exhibits dynamic changes in response to various cellular stimulations:

• Calcium-dependent trafficking:

  • Stimulation with calcium ionophore A23187 (250nM) increases the membrane association of ANXA7

  • Co-localization with lamellar body marker protein ABCA3 increases in a time-dependent manner

  • Co-localization coefficient (CC) with SNAP23 increases from baseline 0.103 to 0.121, 0.200, and 0.295 at 5, 15, and 30 minutes, respectively

• PKC-mediated trafficking:

  • Stimulation with PMA (100nM), a PKC activator, also increases ANXA7 membrane association

  • Co-localization with SNAP23 increases from baseline to 0.129, 0.118, and 0.245 at 5, 15, and 30 minutes, respectively

• Physiological stimulation:

  • Other secretagogues like ATP and terbutaline also promote ANXA7 membrane association

  • ANXA7 trafficking to both plasma membrane and lamellar bodies correlates with surfactant secretion

These observations suggest that ANXA7 plays a role in membrane fusion events during exocytosis, with its trafficking regulated by calcium signaling and protein kinase C pathways. The time-dependent increase in co-localization coefficients indicates progressive recruitment of ANXA7 to specific membrane compartments during stimulated secretion .

What is known about the relationship between ANXA7 and SODD in cancer research?

The relationship between ANXA7 and Suppressor of Death Domains (SODD) in cancer research reveals interesting molecular connections:

• Expression correlation:

  • Down-regulation of ANXA7 leads to significantly decreased expression of SODD protein

  • This suggests that ANXA7 may regulate SODD expression or stability

• Functional implications:

  • Both proteins appear to be involved in regulating tumor cell behavior

  • When ANXA7 is silenced, resulting in decreased SODD levels, tumor cells show:

    • Reduced migration through cell membranes

    • Decreased invasion through Matrigel

    • Slower proliferation rates

• Potential mechanisms:

  • SODD is known to regulate cell death pathways by binding to death domains of TNF receptors

  • ANXA7-SODD axis may represent a novel regulatory pathway in tumor progression

  • The exact molecular mechanism linking these proteins remains to be fully elucidated

These findings suggest that targeting the ANXA7-SODD axis could potentially be explored as a therapeutic strategy in certain cancers, as down-regulation of ANXA7 decreases SODD expression and reduces malignant cellular behaviors .

Why might I observe different molecular weights for ANXA7 in Western blot?

Researchers may observe different molecular weights for ANXA7 in Western blot analyses due to several factors:

• Isoform variation:

  • Multiple isoforms of ANXA7 exist due to alternative splicing

  • The observed molecular weights typically range from 47 to 51 kDa

• Species differences:

  • Human ANXA7 may run at a slightly different molecular weight (approximately 50 kDa) compared to mouse or rat ANXA7 (approximately 47 kDa)

• Post-translational modifications:

  • Phosphorylation, ubiquitination, or other modifications can alter protein migration

  • Stimulus-induced modifications may change the apparent molecular weight

• Technical factors:

  • Different gel systems and running conditions can affect protein migration

  • Sample preparation methods (reducing vs. non-reducing conditions)

  • Buffer composition and pH

To address this variability, researchers should include appropriate positive controls and molecular weight markers. For example, the Proteintech ANXA7 antibody (10154-2-AP) reports observed molecular weights of both 47 kDa and 51 kDa, while the R&D Systems antibody (AF3926) detects bands at approximately 50 kDa in human cells and 47 kDa in rodent cells .

What controls should be included when using ANXA7 antibodies?

When using ANXA7 antibodies, the following controls should be included to ensure experimental validity:

• Positive controls:

  • Validated cell lines or tissues known to express ANXA7, such as:

    • Jurkat, HeLa, or A549 cells for human samples

    • C2C12 cells or mouse lung/brain/heart tissues for murine samples

    • L6 cells or rat brain tissue for rat samples

• Negative controls:

  • Antibody specificity controls:

    • Pre-incubation of antibody with recombinant ANXA7 protein

    • Isotype control antibodies

  • Sample controls:

    • ANXA7 knockdown or knockout samples

    • Tissues known not to express significant ANXA7

• Technical controls:

  • Loading controls for Western blot (β-actin, GAPDH, etc.)

  • Secondary antibody-only controls for immunostaining

  • Non-specific binding controls (protein block)

• Experimental condition controls:

  • For stimulation experiments, include appropriate vehicle controls

  • For time-course studies, include baseline (0 min) samples

Implementing these controls will help validate antibody specificity, confirm ANXA7 detection, and ensure that observed effects are genuinely related to ANXA7 expression or modulation .

How can I optimize immunofluorescence protocols for ANXA7 co-localization studies?

Optimizing immunofluorescence protocols for ANXA7 co-localization studies requires careful consideration of several parameters:

• Fixation optimization:

  • 4% paraformaldehyde for 10 minutes has been validated for ANXA7 detection

  • Avoid over-fixation which can mask epitopes

• Blocking and permeabilization:

  • Simultaneous blocking and permeabilization for 30 minutes

  • Use appropriate detergents (Triton X-100, Tween-20) at concentrations that maintain cellular structure

• Sequential staining strategy for antibodies from the same species:

  • Complete staining for the first antigen (e.g., SNAP23) using standard protocols

  • Use biotin blocking agents (30 min) to prevent cross-reactivity

  • Apply biotinylated ANXA7 antibodies (1:500, overnight)

  • Detect with streptavidin-AlexaFluor568 conjugates (1:500, 1 hour)

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider targeted cellular components when selecting fluorophores

• Imaging parameters:

  • Use confocal laser scanning microscopy for superior optical sectioning

  • Apply appropriate settings to minimize bleed-through between channels

  • Collect Z-stacks for 3D analysis of co-localization

• Quantification methods:

  • Calculate co-localization coefficients (CC)

  • Consider weighted CC values (normalized for total intensity)

  • Use specialized software for co-localization analysis

This optimized approach has successfully demonstrated increased co-localization of ANXA7 with proteins like SNAP23 and ABCA3 in type II cells after stimulation with secretagogues, with time-dependent increases in co-localization coefficients .

What role does ANXA7 play in tumor cell biology and metastasis?

Research into ANXA7's role in tumor cell biology and metastasis has revealed several important findings:

• Regulation of tumor cell behaviors:

  • Down-regulation of ANXA7 significantly reduces tumor cell migration through cell membranes

  • ANXA7 silencing decreases tumor cell invasion through Matrigel

  • Proliferation rate of tumor cells is slower when ANXA7 expression is reduced

• Molecular pathway interactions:

  • ANXA7 appears to regulate expression of SODD (Suppressor of Death Domains)

  • Down-regulation of ANXA7 leads to decreased SODD expression

  • This ANXA7-SODD axis may represent a novel regulatory pathway in tumor progression

• Potential as a therapeutic target:

  • Given its role in promoting migration, invasion, and proliferation, ANXA7 could be explored as a therapeutic target

  • Silencing ANXA7 gene expression could potentially suppress metastatic potential

  • Further research is needed to elucidate the precise mechanisms and potential side effects

These findings suggest that ANXA7 plays a significant role in promoting aggressive tumor cell behaviors that contribute to metastasis. The correlation between ANXA7 expression and these behaviors, as well as its relationship with SODD, positions ANXA7 as an interesting target for further cancer research .

How can ANXA7 be studied in relation to membrane trafficking and exocytosis?

ANXA7 can be studied in relation to membrane trafficking and exocytosis using various methodological approaches:

• Co-localization studies:

  • Immunofluorescence co-localization with membrane markers like SNAP23 and ABCA3

  • Use confocal microscopy to track ANXA7 movement to specific membrane compartments

  • Calculate co-localization coefficients to quantify associations

  • Perform time-course studies after stimulation with secretagogues

• Functional secretion assays:

  • Measure surfactant secretion in type II cells with modulated ANXA7 expression

  • Track vesicle exocytosis using fluorescent markers

  • Combine with calcium imaging to correlate calcium signals with ANXA7 trafficking

• Stimulation protocols:

  • Use calcium ionophores (A23187, 250nM) to trigger calcium-dependent membrane fusion

  • Apply PKC activators (PMA, 100nM) to stimulate secretion

  • Test physiological secretagogues (ATP, terbutaline) to induce exocytosis

• Subcellular fractionation:

  • Isolate plasma membrane and vesicle fractions

  • Analyze ANXA7 distribution before and after stimulation

  • Perform binding assays with purified ANXA7 and membrane fractions

• Advanced imaging techniques:

  • Live-cell imaging to track ANXA7-GFP fusion proteins during exocytosis

  • Super-resolution microscopy for detailed localization

  • TIRF microscopy to visualize membrane-proximal events

Research has demonstrated that ANXA7 shows increased membrane association after stimulation with secretagogues, with time-dependent increases in co-localization with both ABCA3 (a lamellar body marker) and SNAP23. These findings suggest that ANXA7 plays a role in both vesicle trafficking and plasma membrane events during stimulated secretion .

What advanced techniques are being developed for studying ANXA7 interactions?

Advanced techniques for studying ANXA7 interactions are continuously evolving, offering new insights into its biological functions:

• Proximity-based interaction mapping:

  • BioID or TurboID approaches to identify proteins in close proximity to ANXA7

  • Proximity ligation assay (PLA) for visualizing protein-protein interactions in situ

  • APEX2-based proximity labeling for subcellular interaction mapping

• Mass spectrometry-based approaches:

  • Immunoprecipitation coupled with mass spectrometry (IP-MS)

  • Cross-linking mass spectrometry (XL-MS) to capture transient interactions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for structural dynamics

• Advanced microscopy techniques:

  • Förster resonance energy transfer (FRET) to measure protein-protein interactions

  • Fluorescence lifetime imaging microscopy (FLIM) for protein interaction dynamics

  • Single-molecule tracking to follow individual ANXA7 molecules

• Functional genomics approaches:

  • CRISPR-Cas9 screening to identify genetic interactors

  • RNA-seq analysis following ANXA7 modulation

  • Phosphoproteomics to map signaling networks affected by ANXA7

• Structural biology techniques:

  • Cryo-electron microscopy of ANXA7-membrane complexes

  • X-ray crystallography of ANXA7 with binding partners

  • Nuclear magnetic resonance (NMR) for dynamics studies

These emerging techniques offer promising avenues to better understand ANXA7's molecular interactions, membrane-binding properties, and role in various cellular processes. Their application will provide deeper insights into the mechanistic details of how ANXA7 contributes to membrane trafficking, cell signaling, and potentially tumor biology .

How is our understanding of ANXA7 function evolving in different research fields?

Our understanding of ANXA7 function is rapidly evolving across multiple research fields:

• Cell biology:

  • ANXA7 is increasingly recognized as a key player in membrane organization and dynamics

  • Its role in calcium-dependent membrane binding and trafficking continues to be elucidated

  • Evidence supports its function in regulating exocytosis in specialized secretory cells

• Cancer biology:

  • Emerging evidence links ANXA7 to tumor cell migration, invasion, and proliferation

  • The relationship between ANXA7 and SODD suggests novel regulatory pathways

  • Down-regulation of ANXA7 reduces aggressive cancer cell behaviors, highlighting its potential as a therapeutic target

• Pulmonary research:

  • ANXA7's role in surfactant secretion in alveolar type II cells has been established

  • Co-localization with SNAP23 and ABCA3 suggests involvement in both vesicle trafficking and plasma membrane events during exocytosis

  • Time-dependent trafficking in response to secretagogues reveals dynamic regulation

• Methodological advances:

  • Development of specific antibodies has enabled diverse applications across species

  • Advanced imaging and biochemical techniques continue to reveal new aspects of ANXA7 function

  • Molecular tools for modulating ANXA7 expression provide opportunities for functional studies

Future research will likely further integrate these areas, potentially revealing new therapeutic applications and deeper understanding of fundamental cellular processes involving ANXA7 .

What are the most promising research directions for ANXA7 antibody applications?

The most promising research directions for ANXA7 antibody applications include:

• Biomarker development:

  • Exploration of ANXA7 as a diagnostic or prognostic marker in certain cancers

  • Development of high-sensitivity detection methods for clinical applications

  • Correlation of ANXA7 expression patterns with disease progression

• Therapeutic target validation:

  • Antibody-based targeting of ANXA7 for potential cancer therapies

  • Development of antibody-drug conjugates directed against ANXA7

  • In vivo imaging using labeled ANXA7 antibodies to track disease progression

• Mechanism elucidation:

  • Detailed mapping of ANXA7 interactions during membrane trafficking

  • Investigation of ANXA7-SODD regulatory axis in normal and pathological conditions

  • Understanding the calcium-dependent conformational changes in ANXA7

• Technical innovations:

  • Development of phospho-specific antibodies to track ANXA7 activation states

  • Creation of nanobody-based tools for super-resolution imaging

  • Engineering of intrabodies to track and modulate ANXA7 in living cells

• Cross-species comparative studies:

  • Leveraging antibodies that recognize ANXA7 across human, mouse, and rat to investigate conserved functions

  • Understanding species-specific differences in ANXA7 regulation and function

  • Translation of findings from model organisms to human health applications