Anxa4 Antibody
Description
Introduction to Annexin A4 Antibody
Annexin A4 antibodies are immunological reagents specifically designed to detect and bind to Annexin A4 (Anxa4), a member of the calcium- and phospholipid-binding protein family. These antibodies serve as critical tools for investigating Anxa4's diverse biological functions in both normal physiology and pathological states. Commercially available in various formats, these antibodies enable researchers to study Anxa4 through multiple experimental approaches .
Annexin A4, the target protein, belongs to the larger Annexin superfamily. It shares 45-59% sequence identity with other Annexin family members and exhibits calcium-dependent phospholipid-binding properties . The protein is predominantly expressed in epithelial cells of multiple organs, with notable presence in the liver and pancreas . Understanding Anxa4's biological roles has significant implications for cancer research, cell survival mechanisms, and potential therapeutic interventions.
Structure and Characteristics of Anxa4 Antibodies
Anxa4 antibodies are available in multiple formats, each with distinct characteristics suited for specific experimental applications. These antibodies are generated in various host species, with rabbit and mouse being the most common .
Types and Formats
The most common types of Anxa4 antibodies include:
Specificity and Cross-Reactivity
Most commercial Anxa4 antibodies demonstrate high specificity for their target, with minimal cross-reactivity with other proteins. For example, the Human/Mouse/Rat Annexin A4 Antibody from R&D Systems shows no cross-reactivity with recombinant human Annexin proteins in Western blot applications . Species reactivity varies between antibodies, with some recognizing Anxa4 across multiple species (human, mouse, rat) while others are species-specific .
Applications of Anxa4 Antibodies
Anxa4 antibodies are versatile tools employed across multiple experimental techniques for studying protein expression, localization, and function.
Common Laboratory Applications
Optimization Considerations
Successful application of Anxa4 antibodies requires optimization for specific experimental conditions. For IHC applications, antigen retrieval is often necessary, with some antibodies performing optimally with TE buffer pH 9.0 or citrate buffer pH 6.0 . Storage conditions also vary, with most antibodies requiring storage at -20°C, though some can be stored at 4°C for short-term use to avoid freeze-thaw cycles .
Biological Significance of Annexin A4
Research utilizing Anxa4 antibodies has significantly advanced our understanding of this protein's biological roles and disease associations.
Evolutionary Conservation and Expression Pattern
Anxa4 is an evolutionarily conserved protein expressed in multiple tissues. During development, Anxa4 is broadly expressed in the developing liver and pancreas, and later becomes more restricted to the hepatopancreatic ducts and pancreatic islets, including insulin-producing β-cells . This expression pattern is conserved between zebrafish and mammals, suggesting fundamental biological functions across vertebrate species .
Cellular Localization and Function
Anxa4 is often associated with the inner leaflet of the plasma membrane, consistent with its calcium- and phospholipid-binding properties . The protein has been implicated in multiple cellular functions including:
Anxa4 in Cancer Biology
Anxa4 expression is altered in multiple cancer types, making Anxa4 antibodies valuable tools for cancer research.
Mechanistically, Anxa4 appears to exert anti-apoptotic effects in cancer cells by activating NF-κB transcriptional activity, potentially contributing to chemoresistance . These findings have positioned Anxa4 as a potential cancer diagnostic and therapeutic target .
Anxa4 in Diabetes
Anxa4 has emerging connections to diabetes pathophysiology. It is a downstream target of several monogenic diabetes genes, including PDX1, HNF1A, and HNF4A . Given Anxa4's role in pancreatic β-cell development and potentially in cell survival, it may have implications for the mechanism of diabetic β-cell apoptosis .
Developmental Biology Studies
Anxa4 antibodies have been instrumental in characterizing the protein's role in embryonic development. In zebrafish, the monoclonal antibody 2F11 (which targets Anxa4) has been used to study hepatopancreatic development . Through knockdown studies combined with Anxa4 antibody labeling, researchers identified Anxa4 as a critical factor for liver progenitor viability .
Cell Survival Mechanisms
Investigations utilizing Anxa4 antibodies have revealed that Anxa4 inhibits the extrinsic apoptotic pathway. Knockdown of Anxa4 in zebrafish led to elevated expression of caspase 8 and Δ113p53, along with liver bud-specific activation of Caspase 3 and subsequent apoptosis . These findings represent the first in vivo investigation linking Anxa4 to cell viability, consistent with previous in vitro studies .
Cancer Research Applications
Anxa4 antibodies are frequently employed in cancer research to examine expression patterns across different tumor types and correlate these patterns with clinical outcomes. The increased expression of Anxa4 in multiple cancer types has been associated with decreased cell-to-cell adhesion, increased metastatic potential, and resistance to chemotherapy .
Technical Considerations for Anxa4 Antibody Selection
When selecting an Anxa4 antibody for research, several factors should be considered:
Antibody Validation Status
Properly validated antibodies are essential for generating reliable data. Reputable manufacturers typically validate their Anxa4 antibodies through multiple applications including Western blot, IHC, ICC, and ELISA with known positive and negative controls . Researchers should review validation data before selecting an antibody.
Species Reactivity Requirements
Different Anxa4 antibodies show varying cross-reactivity across species. Some antibodies recognize Anxa4 from multiple species (human, mouse, rat), while others are species-specific . Researchers should select antibodies with validated reactivity for their experimental model species.
Application-Specific Considerations
The optimal antibody may vary depending on the intended application. For instance, monoclonal antibodies often provide higher specificity but potentially lower sensitivity compared to polyclonal antibodies. For quantitative applications like ELISA, validated antibody pairs specifically designed for sandwich assays should be selected .
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research findings suggest that AnxA4 directly inhibits the activity of adenylate cyclase 5 (AC5), revealing a novel regulatory function of annexins. PMID: 26023182
- Fhit, a tumor suppressor protein, relocates annexin A4 from the plasma membrane to the cytosol, enhancing the sensitivity of lung cancer cells to paclitaxel. PMID: 24223161
- NME1, a metabolic enzyme, regulates the abundance of annexin IV and EF-1Balpha through post-translational mechanisms. PMID: 21541759
- Studies have investigated intron disruptions in the annexin IV gene that generate novel transcripts. PMID: 12912993
Customer Reviews
Applications : Western Blot
Review: Another protein we validated by Western blot was annexin A4 (ANXA4), which is downregulated upon WA treatment according to the 2D-LC-Orbitrap experiment results.
Q&A
What is ANXA4 and what are its known cellular functions?
ANXA4 is a calcium/phospholipid-binding protein that promotes membrane fusion and is involved in exocytosis . It associates with the inner surface of the plasma membrane and plays critical roles in processes like vesicle trafficking and membrane dynamics . Research has demonstrated ANXA4 localization at the stereociliary tips in hair cells, suggesting potential functions in specialized cellular structures . Additionally, ANXA4 appears to influence cell migration, invasion, and adhesion processes, as evidenced by functional assays with mutant ANXA4 proteins .
What alternative names and protein designations exist for ANXA4?
ANXA4 is known by several alternative designations in scientific literature, including: ANX4, Annexin A4, 35-beta calcimedin, Annexin IV, Carbohydrate-binding protein p33/p41, Chromobindin-4, Endonexin I, Lipocortin IV, P32.5, PP4-X, Placental anticoagulant protein II, and Protein II, PAP-II . Researchers should be aware of these alternative nomenclatures when conducting literature searches or cross-referencing studies.
What applications are ANXA4 antibodies validated for?
ANXA4 antibodies have been validated for multiple laboratory applications, including:
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Western blotting (WB) - typically at 1:1000-1:2000 dilutions
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Immunohistochemistry on paraffin sections (IHC-P) - typically at 1:500 dilution
Each application requires specific optimization protocols, and validation should be performed for the specific research context and tissue/cell type being studied.
How should ANXA4 antibody specificity be validated?
Proper antibody validation is essential for reliable experimental results. For ANXA4 antibodies, specificity can be validated through:
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Knockdown/knockout controls - Multiple studies confirm antibody specificity using ANXA4 knockdown samples with clear band reduction in Western blots
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Expected molecular weight verification - ANXA4 typically appears at 35-37 kDa on Western blots
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Cross-reactivity testing with other annexin family members
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Testing in ANXA4 knockout models - CRISPR/Cas9-generated knockout mice provide excellent negative controls
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Multiple antibodies targeting different epitopes should yield consistent results
What methodological approaches are effective for ANXA4 overexpression studies?
When conducting ANXA4 overexpression experiments, researchers have successfully employed the following methodology:
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Plasmid selection: Full human ANXA4 coding sequence with C-terminal GFP tags has been successfully used in multiple studies
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Transfection technique: Lipofectamine 3000 reagent following manufacturer's protocol has shown good efficiency
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Validation methods: Sequence confirmation, GFP fluorescence imaging, and Western blot analysis provide multi-level verification
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Controls: Empty vector controls containing only the tag should be included to account for tag-related effects
How can ANXA4 knockdown be effectively achieved and validated?
For ANXA4 knockdown experiments, researchers should consider:
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siRNA selection: ON-TARGETplus SMARTpool siRNAs have been successfully used at 20 nM concentration
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Transfection reagent: DHARMAfect 1 Reagent for 48 hours has demonstrated effective knockdown
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Controls: Scrambled non-targeting controls are essential for differentiating specific from non-specific effects
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Validation: Western blotting with ANXA4-specific antibodies (1:2000 dilution) can confirm knockdown efficiency
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Functional validation: Assessing biological consequences of knockdown through relevant functional assays
What protocols are recommended for Western blot analysis of ANXA4?
Optimal Western blot protocols for ANXA4 detection include:
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Protein extraction: RIPA lysis buffer containing protease and phosphatase inhibitors
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Gel electrophoresis: 12% sodium dodecyl sulfate poly-acrylamide gel
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Transfer: Standard transfer to polyvinylidene difluoride membrane
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Loading control: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at 1:10,000 dilution
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Secondary antibody: IRDye secondary antibody at 1:10,000 dilution
How can researchers study ANXA4 localization in specialized cellular structures?
For investigating ANXA4 in specialized cellular structures such as stereocilia:
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Tissue preparation: Whole-mount immunostaining of cochlear tissue has successfully revealed ANXA4 localization
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Antibody selection: Commercial anti-ANXA4 antibodies with verified specificity in knockout models
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High-resolution imaging: Detailed examination of subcellular structures requires appropriate magnification and resolution
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Multiple antibody validation: Using different commercial anti-ANXA4 antibodies to confirm localization patterns
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Knockout controls: ANXA4 knockout tissues provide essential negative controls to confirm antibody specificity
How can ANXA4 mutations be investigated in reproductive pathologies?
For studying ANXA4 mutations in recurrent spontaneous abortion (RSA) and other reproductive disorders:
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Genomic screening approach: Whole-exome sequencing (WES) of patient samples with appropriate controls
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Variant filtering: Use bioinformatic pipelines to filter variants based on frequency in public databases (dbSNP, 1000 Genomes Project, ExAC)
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Validation: Sanger sequencing confirmation of identified variants
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Primer design: Design primers flanking the mutation site (e.g., F: 5'-GGCCTCGAAGAACTTCTGCT-3′, R: 5'-TGGGCATCTTCCATGGCATT-3′ for ANXA4)
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PCR conditions: 94°C for 5 min, followed by 30 cycles (94°C for 30s, 52°C for 30s, 72°C for 30s) and final extension at 72°C for 10 min
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Functional assessment: Cell migration, invasion, and adhesion assays to determine the functional impact of mutations
What approaches can be used to study ANXA4 function in hair cells?
ANXA4 function in cochlear and vestibular hair cells can be investigated through:
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Knockout model generation: CRISPR/Cas9 technique has been successfully used to generate ANXA4 knockout mice
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gRNA design: Targeting genomic sequences such as 5′-TGTTATAAATATAACGCACAAGG-3′ and 5′-AGCCTGAGCCTACACCTCGAGGG-3′
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Genotyping: Using specific primer pairs for wild-type and knockout alleles
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Functional assessments: Evaluation of stereocilia morphology, mechano-electrical transduction (MET), and auditory/vestibular functions
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Immunolocalization: Whole-mount immunostaining to determine ANXA4 distribution within hair cells
How should functional redundancy be addressed in ANXA4 research?
When studying proteins like ANXA4 that may exhibit functional redundancy:
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Comprehensive phenotypic analysis: Despite apparent normal development in ANXA4 knockout mice, detailed examination across multiple parameters is essential
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Investigation of compensatory mechanisms: Assess potential upregulation of other annexin family members in ANXA4 knockout models
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Double/multiple knockout approaches: Generate compound knockouts of ANXA4 with related family members to uncover masked phenotypes
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Stress/challenge conditions: Test knockout models under various stressors that might reveal phenotypes not evident under normal conditions
Comparison of ANXA4 antibodies and applications
*Antibody from Sigma
Known ANXA4 mutations and their functional impacts
ANXA4 knockout effects in different tissue contexts
How can researchers address non-specific binding with ANXA4 antibodies?
Non-specific binding is a common challenge in immunodetection. For ANXA4 antibodies:
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Validation controls: Always include ANXA4 knockdown or knockout samples as essential negative controls
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Blocking optimization: 5% bovine serum albumin has shown good results in reducing background
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Antibody titration: Test multiple dilutions to find optimal signal-to-noise ratio
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Cross-reactivity assessment: Test antibodies against recombinant annexin family members to ensure specificity
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Pre-absorption controls: Pre-incubate antibodies with recombinant ANXA4 to confirm binding specificity
What strategies can improve detection of low-abundance ANXA4?
For improved detection of ANXA4 in challenging samples:
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Protein enrichment: Consider immunoprecipitation to concentrate ANXA4 before analysis
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Loading optimization: Higher protein amounts (30 μg for whole cell lysates) have been successfully used
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Signal amplification: Consider enhanced chemiluminescence systems for Western blot or tyramide signal amplification for IHC/IF
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Subcellular fractionation: Enrich for membrane fractions where ANXA4 is predominantly localized
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Detection system optimization: Infrared imaging systems have shown good sensitivity for ANXA4 detection
What emerging technologies show promise for ANXA4 research?
Several cutting-edge approaches offer new possibilities for ANXA4 research:
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Single-cell transcriptomics: For cell-specific expression analysis across diverse tissues and conditions
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CRISPR activation/inhibition: For more nuanced functional studies than complete knockouts
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Super-resolution microscopy: For detailed analysis of ANXA4 subcellular localization, particularly in specialized structures like stereocilia
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Patient-derived organoids: For studying ANXA4 mutations in physiologically relevant 3D tissue models
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Multi-omics integration: Combining genomics, proteomics, and functional data for comprehensive understanding of ANXA4 biology
What are promising research areas involving ANXA4?
Based on current literature, several promising research directions emerge:
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Reproductive biology: Further investigation of ANXA4 mutations in pregnancy complications and implantation
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Membrane repair mechanisms: Exploration of ANXA4's role in cellular responses to membrane damage
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Sensory biology: Despite normal development in knockout mice, investigating potential roles under stress conditions or aging in auditory system
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Therapeutic targeting: Exploring ANXA4 as a potential therapeutic target or biomarker in conditions where it shows functional significance
