ANXA10 Antibody

What is ANXA10 and what are its basic cellular functions?

ANXA10 functions as a calcium-dependent phospholipid-binding protein that regulates cell proliferation and differentiation. It is the latest identified member of the annexin family of proteins . ANXA10 plays important roles in:

• Calcium-dependent phospholipid binding

• Cell cycle regulation, particularly in promoting G1 phase progression

• Cell migration and invasive capacity in certain cancer types

• Tumor differentiation processes in several cancers

What types of ANXA10 antibodies are available for research applications?

Several types of validated ANXA10 antibodies are available for research:

• Rabbit polyclonal antibodies (e.g., ab227556, HPA074650) suitable for Western blot, IHC-P with human samples

• Rabbit monoclonal antibodies (e.g., NBP1-90156) used in knockout validation studies

• Custom-developed antibodies used in specialized research (e.g., those developed by Dr. MP Fernández for HNSCC studies)

Different antibodies are validated for specific applications including WB, IHC-P, and ICC-IF, with varying degrees of species reactivity .

What are the recommended protocols for immunohistochemical detection of ANXA10?

For optimal IHC detection of ANXA10 in formalin-fixed, paraffin-embedded tissue:

• Cut tissue into 3-μm sections and dry on IHC microscope slides

• Deparaffinize with xylene and hydrate through graded alcohols

• Perform antigen retrieval with proteinase K

• Block endogenous peroxidase with 3% hydrogen peroxide for 15 min

• Incubate with primary antibody at 1:100 concentration for 45 min (optimal concentration may vary by antibody)

• Apply immunodetection system (e.g., Dako EnVision Flex + Visualization System)

• Develop with diaminobenzidine as chromogen

• Counterstain with hematoxylin for 7 min

• Dehydrate and mount slides

For standardized scoring, a semiquantitative system based on staining intensity is recommended, with scores of negative (0), weak to moderate (1+), and strong positive (2+) .

How should I design experiments to study ANXA10's role in cancer cell behavior?

Based on recent studies, a comprehensive approach would include:

Experimental design elements:

• Expression analysis:

  • Compare ANXA10 expression between tumor and normal tissues using IHC

  • Correlate with clinicopathological parameters including tumor grade, stage, and patient outcomes

• Functional studies:

  • Generate ANXA10 knockout cell lines using CRISPR/Cas9 (as demonstrated in gastric cancer studies)

  • Alternatively, use siRNA for transient knockdown (as shown in lung adenocarcinoma studies)

  • Assess phenotypic changes in:

    • Proliferation (using CCK-8 or similar assays)

    • Migration (using wound healing assays)

    • Invasion (using Transwell assays)

    • Cell cycle progression (using flow cytometry)

• Mechanistic studies:

  • Analyze downstream pathways using transcriptomic approaches

  • Validate key targets with qRT-PCR and Western blot

  • Focus on cell cycle regulatory proteins (particularly G1/S checkpoint proteins)

For optimal results, include multiple cell lines representing your cancer type of interest and validate findings with patient samples whenever possible .

For investigating ANXA10's calcium-dependent binding properties:

• Liposome binding assays:

  • Prepare recombinant ANXA10 protein (bacterial expression using pET-23a vector system is documented)

  • Incubate purified protein with binding buffer containing different Ca² concentrations (0.5, 1, 2, or 5 mM CaCl₂, with 1 mM EGTA as a control)

  • Use brain extract liposomes at 2 mg/ml final concentration

  • Incubate for 1 hour at room temperature

  • Pellet liposomes at 100,000g for 30 min at 4°C

  • Wash pellet in binding buffer and analyze by SDS-PAGE with Coomassie staining

  • Quantify signal intensities by densitometric analysis

• Phosphoinositide binding studies:

  • Block PIP Strips with 1% nonfat-dry milk in PBS (1 hour, room temperature)

  • Add purified ANXA10 at 2 μg/ml final concentration

  • Incubate overnight at 4°C in blocking buffer containing either 50 or 500 μM Ca²⁺

  • Wash with PBS containing 0.1% Tween 20

  • Maintain respective Ca²⁺ concentrations during all incubation steps

  • Detect bound ANXA10 using anti-ANXA10 antibody via immunoblotting

Statistical analysis should include repeated measures one-way ANOVA followed by Dunnett's multiple comparison test, with p<0.05 indicating significance .

How reliable is ANXA10 as a prognostic marker in different cancers, and what scoring methodologies should be used?

The reliability of ANXA10 as a prognostic marker varies by cancer type:

Scoring methodology for ANXA10 IHC in lung cancer:

• Evaluate expression intensity (negative, weak, moderate, strong)

• Consider both intensity and percentage of positive cells

• Use multivariate analysis to confirm independence from other prognostic factors (pT stage, pN stage, pleural infiltration, vascular and lymphatic invasion)

Recommended scoring approach for research:

• Use a semiquantitative scoring system based on staining intensity (0=negative, 1+=weak/moderate, 2+=strong)

• Always correlate with clinicopathological parameters

• Perform both univariate and multivariate analyses

• Consider combined analysis with other annexin family members (ANXA9 and ANXA10 expression were significantly correlated, Spearman coefficient 0.459, p<0.001)

What mechanisms explain ANXA10's role in chemosensitivity, particularly to 5-FU treatment?

ANXA10 appears to influence chemosensitivity through cell cycle regulation and proliferation mechanisms:

Research investigating these mechanisms should focus on cell cycle analysis, G1/S checkpoint proteins, and comprehensive transcriptomic profiling .

How do I optimize ANXA10 antibody use for detecting nuclear localization versus cytoplasmic expression?

ANXA10 has been observed in both cytoplasmic and nuclear localizations, with potentially different functional implications. To optimize detection of different subcellular localizations:

For optimal nuclear detection:

• Fixation protocol:

  • Use 4% paraformaldehyde (PFA) + 0.1% Triton X-100 in PBS for 3 minutes

  • Follow with 4% PFA alone for 10 minutes at room temperature

  • This two-step fixation enhances nuclear membrane permeability while preserving structure

• Antibody selection and dilution:

  • For paraspeckle co-localization studies, pair anti-ANXA10 with anti-SFPQ antibodies

  • Optimize antibody concentration (typically 1:500-1:1000 for nuclear detection)

  • Extend primary antibody incubation to overnight at 4°C

• Visualization techniques:

  • For co-localization studies, use confocal microscopy (e.g., LSM 510 META or LSM 780)

  • Employ a high-quality oil immersion objective (Plan-Apochromat ×63/1.4)

  • Use nuclear counterstain like DRAQ5

For membrane/cytoplasmic fraction analysis:

• Cell fractionation protocol:

  • Hypotonic lysis followed by differential centrifugation

  • Collect membrane pellet after centrifugation at high speed (≥100,000g)

  • Resuspend in 2× SDS sample buffer for Western blot analysis

• Controls and normalization:

  • Include membrane markers (e.g., caveolin) and cytosolic markers (e.g., GAPDH)

  • Normalize ANXA10 signal to appropriate compartment markers

  • Quantify by densitometric analysis

• Verification approaches:

  • Complement IHC/ICC with subcellular fractionation and Western blot

  • For studies of translocation, induce with relevant stimuli (e.g., 5 μM doxorubicin has been used to study nuclear localization)

What is the relationship between ANXA10 and other annexin family members in experimental settings?

The relationship between ANXA10 and other annexin family members presents important experimental considerations:

• Co-expression patterns:

  • ANXA9 and ANXA10 expression were significantly correlated in HNSCC (Spearman correlation coefficient 0.459, p<0.001)

  • This suggests potential functional relationships or shared regulatory mechanisms

• Experimental approaches to study annexin family relationships:

  • Comparative expression analysis: Examine multiple annexins simultaneously in the same tissue samples

  • Co-immunoprecipitation: Determine physical interactions between annexin family members

  • GFP fusion constructs: Compare subcellular localization patterns (anxA1GFP, anxA2GFP, and anxA10GFP constructs have been described)

  • mCherry constructs: For co-localization studies with multiple annexins (e.g., anxA2-mCherry)

• Functional redundancy testing:

  • Test whether other annexins can compensate for ANXA10 loss

  • Design experiments with multiple knockdowns/knockouts

  • Compare phenotypic effects between single and combined annexin alterations

• Structural and functional distinctions:

  • Despite family similarities, ANXA10 has unique calcium-binding properties

  • Unlike other annexins, ANXA10 lacks the canonical type II calcium-binding site in domain 1

  • This affects membrane binding properties and should be considered in experimental designs

How should I design ANXA10 knockout or knockdown experiments to study its function in cancer cells?

Based on successful studies, here is a comprehensive approach to ANXA10 functional studies through genetic manipulation:

CRISPR/Cas9 knockout approach:

• Design and validation:

  • Design guide RNAs targeting ANXA10 exons (early exons preferred)

  • Include non-targeting control guide RNAs

  • Validate knockout by Western blotting using reliable antibodies (e.g., NBP1-90156, 1:500)

• Functional assays after knockout:

  • Proliferation: CCK-8 assay at 24h, 48h, 72h, and 96h timepoints

  • Invasion: Transwell invasion assays with Matrigel coating

  • Migration: Wound healing assay with monitoring at 0h, 6h, and 12h

  • Chemosensitivity: Test multiple concentrations of chemotherapeutics (particularly 5-FU)

siRNA knockdown approach:

• Transfection and verification:

  • Transfect cells with ANXA10-specific siRNA and negative control

  • Verify knockdown efficiency by qRT-PCR and Western blot

  • Optimal knockdown usually achieved 48-72h post-transfection

• Cell cycle analysis:

  • Flow cytometry to determine cell cycle distribution

  • Western blot for cell cycle proteins including:

    • Cyclins (D1, E)

    • Cyclin-dependent kinases

    • Checkpoint proteins

• In vivo validation:

  • Xenograft models using knockout/knockdown cells

  • Measure tumor growth rates and final tumor weight/volume

  • Perform IHC on tumor sections to confirm maintained knockout/knockdown

What are the latest transcriptomic findings regarding ANXA10's role in cancer progression?

Recent transcriptomic analyses have revealed important insights into ANXA10's role in cancer progression:

• Gastric cancer transcriptomic findings:

  • ANXA10 knockout in gastric cancer cell lines revealed several candidate pathways and genes regulated by ANXA10

  • Key genes identified include:

    • Claudin 1 (CLDN1) - involved in tight junction formation

    • Keratin 80 (KRT80) - epithelial structural protein

    • RANBP2-type and C3HC4-type zinc finger containing 1 (RBCK1) - regulator of NF-κB signaling

    • Solute carrier family 7 member 5 (SLC7A5) - amino acid transporter

• Lung adenocarcinoma findings:

  • Combined transcriptome sequencing and TCGA data analysis identified ANXA10 as one of the most significant differentially expressed genes

  • ANXA10 was identified as a cell cycle regulation gene in lung adenocarcinoma

  • Knockdown studies showed it affects G1/S phase transition

  • ANXA10 knockdown inhibited the expression of cyclin E, a key cell cycle checkpoint protein

• Integrated multi-omics approaches:

  • Studies combining self-transcriptome sequencing with TCGA data analysis provide robust identification of ANXA10's significance

  • Validation at both mRNA and protein levels confirmed ANXA10's role in cancer progression

  • In vivo tumor formation assays showed decreased tumor formation ability after ANXA10 knockdown

• Research limitations and future directions:

  • Sample size limitations in RNA-seq data

  • Differences in clinical characteristics between TCGA and self-sequencing samples

  • Need for further exploration of molecular mechanisms

  • Potential for ANXA10 as a therapeutic target

The current transcriptomic evidence suggests ANXA10 functions primarily through cell cycle regulation and may be a valuable therapeutic target, particularly in lung adenocarcinoma and gastric cancer .

What are the most common technical issues when working with ANXA10 antibodies and how can they be resolved?

Common technical issues and their solutions when working with ANXA10 antibodies include:

Western blotting issues:

• Multiple bands or unexpected band size:

  • Expected ANXA10 band size: 37 kDa

  • Solution: Use positive control samples (e.g., HCT 116 human colorectal carcinoma cell line)

  • Validate specificity through knockout/knockdown controls

  • Optimize antibody concentration (typical working dilution: 1/1000)

• Weak signal:

  • Increase protein loading (30 μg whole cell lysate recommended)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced chemiluminescence detection systems

  • Ensure proper transfer efficiency

Immunohistochemistry challenges:

• High background:

  • Optimize blocking (2% BSA in PBS for 15 min recommended)

  • Reduce primary antibody concentration (test dilution series: 1/100 to 1/500)

  • Extend washing steps

  • Include appropriate negative controls (omit primary antibody)

• Weak or variable staining:

  • Optimize antigen retrieval (proteinase K recommended)

  • Standardize fixation protocols (fixation time affects epitope accessibility)

  • Ensure consistent section thickness (3-μm recommended)

  • Use sensitive detection systems (e.g., Dako EnVision Flex + Visualization System)

• Scoring variability:

  • Implement standardized scoring system (0=negative, 1+=weak/moderate, 2+=strong)

  • Use multiple independent observers for scoring

  • Include positive controls (vascular endothelium has been used)

Verification approaches:

• Confirm antibody specificity through multiple applications (WB, IHC, ICC)

• Use alternative antibodies targeting different epitopes

• Perform peptide competition assays to confirm specificity

• Include tissue with known expression patterns as controls

How can I validate ANXA10 antibody specificity in my experimental system?

A comprehensive validation strategy for ANXA10 antibodies should include:

• Genetic validation approaches:

  • Generate ANXA10 knockout cell lines using CRISPR/Cas9

  • Use siRNA knockdown to create transient ANXA10 reduction

  • Verify absence or reduction of signal in these negative controls by Western blot

• Recombinant protein controls:

  • Use recombinant ANXA10 protein as a positive control

  • Generate bacterial expression constructs (e.g., pET-23a vector system has been documented)

  • Perform peptide competition assays where antibody is pre-incubated with recombinant ANXA10

• Multi-technique validation:

  • Confirm concordance across different techniques:

    • Western blot (expected band at 37 kDa)

    • IHC (comparing expression patterns across tissues)

    • Immunofluorescence (subcellular localization)

  • Different techniques provide complementary validation

• Tissue/cell expression panels:

  • Test antibody on tissues with known ANXA10 expression patterns

  • Gastric tissue has been used as a reliable positive control

  • Vascular endothelium has also been used as internal positive control

  • Compare expression patterns with published data and public databases

• Fusion protein approaches:

  • Generate ANXA10-GFP fusion constructs (pEGFP-N3 vector system has been used)

  • Verify co-localization of antibody signal with GFP signal

  • This confirms antibody recognition of the target protein in cellular context

• Alternative antibody comparison:

  • Test multiple antibodies targeting different ANXA10 epitopes

  • Compare staining patterns and signal intensities

  • Consistent results across different antibodies support specificity

Proper validation should be performed for each new experimental system and application to ensure reliable results.

What are the key considerations for reproducing published ANXA10 research findings?

To successfully reproduce published ANXA10 research findings, consider these critical factors:

• Antibody selection and validation:

  • Use the same antibody clone/catalog number when possible

  • If different antibody is used, perform thorough validation

  • Document antibody details including:

    • Source/vendor (e.g., Abcam, Novus Biologicals, Atlas Antibodies)

    • Clone/catalog number (e.g., ab227556, NBP1-90156, HPA074650)

    • Working dilution (typically 1:100-1:1000 depending on application)

• Cell lines and tissue samples:

  • Match cell lines used in original research

  • Document passage number and culture conditions

  • For tissue work, ensure similar fixation protocols

  • Consider tumor heterogeneity in clinical samples

• Experimental protocols:

  • For IHC: Follow detailed protocols including:

    • Section thickness (3-μm recommended)

    • Antigen retrieval method (proteinase K)

    • Incubation times and temperatures

    • Detection systems (e.g., Dako EnVision Flex)

  • For functional studies:

    • Match knockdown/knockout methodology

    • Use same timepoints for assays (e.g., 24h, 48h, 72h for proliferation)

    • For chemosensitivity tests, use identical drug concentrations

• Data analysis and interpretation:

  • Use same scoring systems for IHC (0, 1+, 2+ scale commonly used)

  • Apply consistent statistical methods:

    • For survival analysis: Kaplan-Meier with log-rank test

    • For correlation with clinicopathological factors: Chi-square or Fisher's exact test

    • For multiple comparisons: ANOVA with appropriate post-hoc tests

• Potential sources of variation:

  • Antibody lot-to-lot variation

  • Cell line genetic drift

  • Differences in laboratory reagents and equipment

  • Subtle variations in technique execution

  • Differences in clinical sample characteristics

• Reporting standards:

  • Document all methodological details

  • Report positive and negative controls

  • Include representative images

  • Provide complete statistical analysis

  • Acknowledge limitations and potential confounding factors