ALA8 Antibody

What is Annexin A8 and what are its key characteristics in human tissues?

Annexin A8 is a protein detected in human tissues including placenta, where it's specifically localized to endothelial cells . It has a molecular weight of approximately 36 kDa when analyzed by Western blot under reducing conditions . For researchers studying this protein, understanding its tissue-specific expression is fundamental for experimental design. The protein corresponds to amino acids Ala2-Pro327 of human Annexin A8 (Accession # P13928) .

What are the established methods for detecting Annexin A8 in biological samples?

Annexin A8 can be detected through several validated methods:

• Western Blot: Using 2 μg/mL of Sheep Anti-Human Annexin A8 Antigen Affinity-purified Polyclonal Antibody followed by HRP-conjugated anti-sheep secondary antibody. This approach reliably detects a specific band at approximately 36 kDa in cell lysates such as A549 human lung carcinoma cell line .

• Immunohistochemistry: Effective visualization in paraffin-embedded tissue sections using 1 μg/mL of anti-Annexin A8 antibody with overnight incubation at 4°C, following heat-induced epitope retrieval. Visualization can be achieved using HRP-DAB staining systems with hematoxylin counterstaining .

What controls are essential when using antibodies for protein detection?

Based on established methodologies, researchers should include several controls:

For immunocytochemistry:

• Omission of primary antibodies to assess non-specific binding

• Immunoadsorption of primary antibodies with target antigens

• Replacement of primary antibodies with unrelated IgG from the same or different species

• Omission or replacement of secondary antibodies

For Western blot:

• Positive control samples with known expression of the target protein

• Internal normalization control (e.g., β-actin)

• Primary and secondary antibody controls to validate specificity

These controls are critical for verifying antibody specificity and ensuring reliable interpretation of results.

How should antibody dilutions be optimized for different experimental applications?

Optimal antibody dilutions should be determined by each laboratory for each specific application . The optimization process involves:

• Testing a range of antibody concentrations to identify the optimal signal-to-noise ratio

• For Western blot: Starting with 2 μg/mL for anti-Annexin A8 antibodies and adjusting based on signal strength

• For immunohistochemistry: Starting with 1 μg/mL for paraffin-embedded sections with overnight incubation at 4°C

• Evaluating background staining at each concentration

• Determining the minimum concentration that yields reproducible and specific signal

The optimization should be performed using positive control samples with known expression levels of the target protein.

What protocols yield optimal results for Annexin A8 detection by Western blot?

For optimal detection of Annexin A8 by Western blot:

• Prepare cell lysates under reducing conditions

• Load approximately 25 μg protein per lane along with pre-stained molecular weight markers

• Transfer proteins to PVDF membrane

• Block with appropriate blocking buffer

• Probe with 2 μg/mL of Anti-Human Annexin A8 Antibody

• Follow with HRP-conjugated Anti-Sheep IgG Secondary Antibody

• Develop using chemiluminescence detection systems

• Include β-actin detection for normalization

This methodology consistently detects Annexin A8 at approximately 36 kDa in appropriate samples.

How can researchers design time-course experiments to study protein expression changes?

Based on established experimental approaches, time-course experiments should include:

• Multiple time points (e.g., 24h, 48h, 96h) with appropriate controls for each point

• Consistent cell numbers and culture conditions across all time points

• Multiple replicates (minimum triplicates) per time point

• Normalized quantification methods (e.g., to β-actin for Western blot or per 10^5 cells for secreted proteins)

• Statistical analysis appropriate for time-course data

This table illustrates a typical time-course experimental design for protein expression analysis:

How should researchers perform quantitative analysis of immunofluorescence staining?

Quantification of immunofluorescence staining requires systematic image acquisition and analysis:

• Capture non-overlapping images from the entire area using confocal laser scanning microscopy

• Identify immunopositive cells using optimized detection parameters

• Perform image analysis to estimate immunopositive area fraction (in percent)

• Use cell nuclei counterstained with DAPI for cell counting and normalization

• Analyze multiple fields (minimum 8 per sample) to account for heterogeneity

The analysis should use pre-calibrated computer-assisted digital image analysis systems with consistent parameters across all samples and conditions.

What statistical approaches are appropriate for analyzing antibody-based detection data?

Based on established methodologies, appropriate statistical approaches include:

• Non-parametric tests such as Kruskal-Wallis test followed by rank sums Wilcoxon test for comparing staining intensities or protein levels between groups

• Data presentation as median values with ranges rather than means with standard deviations when data may not be normally distributed

• Setting probability level (P = 0.05) as the limit of significance

• Using specialized statistical software (e.g., SPSS) for complex analyses

This approach is particularly suitable for immunological data, which often does not follow normal distribution patterns.

How can densitometric analysis of Western blots be standardized for reproducible results?

Semi-quantitative densitometric analysis of Western blots requires:

• Digital image capture under non-saturating conditions

• Measurement of integrated optical densities for each target band

• Normalization to housekeeping proteins (e.g., β-actin)

• Calculation of relative expression using the formula:
  $\text{Relative expression} = \frac{\text{Integrated density of target protein}}{\text{Integrated density of β-actin}}$

• Comparison between samples using appropriate statistical tests

This standardized approach enables reliable comparison of protein expression levels across different experimental conditions.

How can antibodies be employed to study co-localization of Annexin A8 with other proteins?

Co-localization studies require:

• Dual immunofluorescence staining using antibodies from different species:

  • Anti-Annexin A8 antibody (e.g., Sheep IgG)

  • Antibody against the protein of interest (e.g., Mouse or Rabbit IgG)

• Species-specific secondary antibodies with distinct fluorophores

• Confocal microscopy imaging with appropriate channel separation

• Analysis of co-localization using specialized software to calculate:

  • Pearson's correlation coefficient

  • Manders' overlap coefficient

  • Co-localization percentage

This methodology enables precise spatial analysis of protein interactions in cellular contexts.

What considerations are important when designing immunoprecipitation experiments with Annexin A8 antibodies?

For successful immunoprecipitation of Annexin A8:

• Antibody selection: Use affinity-purified antibodies with demonstrated specificity

• Lysate preparation: Optimize lysis buffers to maintain protein-protein interactions

• Pre-clearing: Remove non-specifically binding proteins from lysates

• Immunoprecipitation conditions:

  • Antibody amount (typically 2-5 μg per sample)

  • Incubation time and temperature (overnight at 4°C)

  • Washing stringency to remove non-specific interactions

• Detection: Western blot analysis of immunoprecipitated complexes using antibodies against potential interacting partners

These considerations help ensure specific isolation of Annexin A8 protein complexes for interaction studies.

How can researchers approach troubleshooting when Annexin A8 antibodies show unexpected results?

When troubleshooting unexpected antibody results, researchers should systematically evaluate:

• Antibody specificity:

  • Verify recognition of recombinant protein

  • Confirm appropriate molecular weight detection

  • Validate with multiple antibodies against different epitopes

• Sample preparation issues:

  • Protein degradation

  • Insufficient antigen retrieval

  • Masking of epitopes by protein modifications

• Technical parameters:

  • Antibody concentration optimization

  • Incubation conditions adjustment

  • Detection system sensitivity

• Biological variables:

  • Cell/tissue-specific expression levels

  • Post-translational modifications affecting epitope recognition

  • Splice variants or protein isoforms

A systematic approach to troubleshooting ensures reliable and reproducible antibody-based detection.

How can Annexin A8 antibodies be utilized in high-throughput screening approaches?

For high-throughput applications:

• Antibody microarrays:

  • Immobilization of anti-Annexin A8 antibodies on microarray slides

  • Detection of Annexin A8 in multiple samples simultaneously

  • Quantification using fluorescent or chemiluminescent detection

• Automated immunohistochemistry/immunofluorescence:

  • Standardized staining protocols on automated platforms

  • Digital image acquisition and analysis

  • Machine learning algorithms for pattern recognition

• Flow cytometry:

  • Intracellular staining for Annexin A8

  • Multi-parameter analysis with other markers

  • High-throughput single-cell analysis

These approaches enable efficient screening of Annexin A8 expression across multiple samples or conditions.

What methodological considerations are important when validating antibodies for detecting post-translational modifications of Annexin A8?

Validation of antibodies for post-translational modifications requires:

• Specificity validation:

  • Testing against modified and unmodified recombinant proteins

  • Competition assays with modified and unmodified peptides

  • Correlation with mass spectrometry data

• Experimental validation:

  • Treatment with modification-inducing stimuli

  • Treatment with inhibitors of specific modifications

  • Correlation with functional outcomes

• Technical considerations:

  • Sample preparation methods that preserve modifications

  • Appropriate blocking and washing conditions

  • Validation across multiple experimental systems

This rigorous validation ensures reliable detection of specific post-translational modifications that may regulate Annexin A8 function.

How can researchers integrate antibody-based detection with other analytical techniques for comprehensive analysis of Annexin A8?

Comprehensive analysis benefits from integrating multiple techniques:

• Combine antibody-based detection with:

  • Mass spectrometry for precise identification of modifications and interacting partners

  • RNA sequencing for correlation with transcript levels

  • Functional assays to correlate expression with biological activities

• Implement multi-omics integration approaches:

  • Correlation analyses between protein, transcript, and functional data

  • Pathway analysis incorporating protein interaction networks

  • Systems biology approaches to model Annexin A8 function in cellular contexts

• Utilize complementary imaging techniques:

  • Super-resolution microscopy for detailed localization

  • Live-cell imaging for dynamic studies

  • Electron microscopy for ultrastructural localization

This integrated approach provides comprehensive understanding of Annexin A8 biology beyond what any single technique can offer.