ALDH8A1 Antibody

How do I select the appropriate ALDH8A1 antibody for my research?

When selecting an ALDH8A1 antibody, researchers should consider several factors including:

• The specific application (immunohistochemistry, Western blot, immunofluorescence, or ELISA)

• Species reactivity requirements

• Antibody type (monoclonal vs. polyclonal)

• Validation data availability

For example, the ALDH8A1 antibody described in source is a polyclonal antibody suitable for immunohistochemistry (dilution 1:200-1:500) and Western blot applications with verified human reactivity . For broader applications including immunoprecipitation and immunofluorescence, the mouse monoclonal IgG2a antibody described in source offers versatility with reactivity against mouse, rat, and human ALDH8A1 . Always examine the validation data and consider whether the antibody has been verified through enhanced validation methods such as orthogonal RNAseq.

What is the recommended protocol for ALDH8A1 immunohistochemistry?

Based on established immunohistochemical procedures, the following protocol is recommended:

• Prepare 4 μm tissue sections from your specimens

• Use an appropriate staining system (e.g., LSAB+ System-HRP)

• For ALDH8A1 detection, dilute the primary antibody to 1:200-1:500

• Include appropriate controls:

  • Negative control: Omit the primary antibody

  • Isotype-matched control antibody

  • Positive control: Human liver sections are recommended

After staining, scoring can be performed on a scale from 0 (no expression) to 3 (strong tumor-cell staining) . For optimal specificity with ALDH8A1 polyclonal antibodies, ensure they have been affinity isolated and stored appropriately in buffered aqueous glycerol solution at -20°C .

How can I validate the specificity of my ALDH8A1 antibody?

Validating antibody specificity is crucial for generating reliable data. For ALDH8A1 antibodies, implement these validation approaches:

• Enhanced validation methods:

  • Orthogonal RNAseq validation: Compare protein expression detected by the antibody against mRNA expression levels

  • Independent antibody validation: Use multiple antibodies targeting different epitopes to confirm consistent staining patterns

• Technical controls:

  • Include isotype-matched control antibodies

  • Use appropriate positive control tissues (e.g., human liver)

  • Consider knockdown/knockout validation if accessible

• Immunogen sequence verification:

  • Confirm the immunogen sequence used to generate the antibody (e.g., "LPCSSYIDSYDPSTGEVYCRVPNSGKDEIEAAVKAAREAFPSWSSRSPQERSRVLNQVADLLEQS") to ensure specificity for the target protein

What are the optimal storage conditions and handling procedures for ALDH8A1 antibodies?

To maintain antibody integrity and performance:

• Store ALDH8A1 antibodies at -20°C when not in use

• Ship the antibody on wet ice during transport

• Avoid repeated freeze-thaw cycles

• For polyclonal antibodies in buffered aqueous glycerol solution, ensure proper mixing before use

• Follow manufacturer-specific recommendations for each antibody preparation

Proper storage is essential as antibody degradation can lead to decreased sensitivity, increased background, and inconsistent results across experiments.

What potential cross-reactivity issues should I be aware of when using ALDH8A1 antibodies?

When working with ALDH8A1 antibodies, consider these potential cross-reactivity issues:

• Cross-reactivity with other ALDH family members, particularly those with high sequence homology

• The ALDH family comprises 19 functional genes in humans, with several members sharing similar structural domains

• Potential cross-reactivity with ALDH1A1 and ALDH1A3, which are frequently studied in similar contexts (particularly cancer research)

To minimize cross-reactivity concerns:

• Use antibodies that have undergone enhanced validation

• Include appropriate controls in each experiment

• Consider using multiple antibodies targeting different epitopes to confirm findings

How can ALDH8A1 antibodies be used to investigate the kynurenine pathway in tryptophan metabolism?

Recent biochemical evidence has established ALDH8A1's role in the kynurenine pathway. Researchers can use ALDH8A1 antibodies to:

• Map enzyme distribution:

  • Determine tissue-specific expression patterns of ALDH8A1 in relation to other kynurenine pathway enzymes

  • Correlate expression with pathway activity

• Investigate pathway dynamics:

  • Examine ALDH8A1 expression changes under various physiological and pathological conditions

  • Study co-localization with upstream enzymes like ACMSD and downstream metabolites

• Functional studies:

  • Use the antibodies in immunoprecipitation assays to isolate ALDH8A1 for enzymatic activity studies

  • Combine with metabolite analysis to correlate enzyme expression with 2-aminomuconic acid (2-AM) production

The NMR spectroscopy data confirms that ALDH8A1 catalyzes the NAD⁺-dependent oxidation of 2-AMS with catalytic efficiency equivalent to that of AMSDH from Pseudomonas fluorescens, resulting in 2-AM production that spontaneously deaminates and tautomerizes .

What is the significance of ALDH8A1 in cancer research, particularly hepatocellular carcinoma?

ALDH8A1 has emerged as a significant prognostic marker in hepatocellular carcinoma (HCC) research:

Researchers can use ALDH8A1 antibodies to validate gene expression data at the protein level and correlate with clinical outcomes in tumor specimens.

How can I optimize ALDH8A1 antibody use for multiple detection methods in the same experiment?

For comprehensive analysis using multiple detection methods:

• Sequential or multiplexed approaches:

  • For tissue sections: Perform immunohistochemistry followed by careful antibody stripping before immunofluorescence

  • For cells: Consider fixation protocols compatible with both immunofluorescence and subsequent protein extraction for Western blotting

• Antibody selection considerations:

  • Choose conjugated antibodies (HRP, PE, FITC, or Alexa Fluor® conjugates) appropriate for each method

  • Ensure the selected antibody has been validated for all intended applications

  • Consider using the same antibody clone across methods when possible

• Optimization strategy:

  • Begin with manufacturer-recommended dilutions for each technique

  • Perform titration experiments to determine optimal concentration for each application

  • Validate signal specificity independently for each detection method

What are common issues when using ALDH8A1 antibodies for immunohistochemistry and how can they be resolved?

How do I interpret ALDH8A1 localization patterns in immunofluorescence studies?

When interpreting ALDH8A1 localization:

• Expected patterns:

  • Primary cytoplasmic localization is expected based on the enzyme's functional role

  • Potential punctate staining may indicate association with specific organelles

• Co-localization considerations:

  • Consider co-staining with markers for subcellular compartments

  • For retinal metabolism studies: Co-stain with retinoid X receptors

  • For kynurenine pathway studies: Co-stain with other pathway enzymes

• Quantification approaches:

  • Measure intensity throughout the cell and calculate cytoplasmic-to-nuclear ratio

  • For cancer studies, correlate expression patterns with clinical parameters

  • Consider automated image analysis to reduce subjective interpretation

How can I apply ALDH8A1 antibodies in functional enzyme activity studies?

To correlate ALDH8A1 protein expression with enzymatic activity:

• Combined immunodetection and activity assays:

  • Use immunoblotting to quantify protein levels

  • Perform parallel enzyme activity assays using the 2-hydroxymuconate semialdehyde (2-HMS) substrate analog

  • Compare results to establish protein-activity correlation

• Immunoprecipitation-based activity assays:

  • Use ALDH8A1 antibodies to immunoprecipitate the enzyme from cell lysates

  • Perform activity assays on the immunoprecipitated protein

  • Include controls to verify specificity of the pull-down

• Monitoring reaction products:

  • Track the NAD⁺-dependent oxidation reaction spectrophotometrically

  • The oxidized product (2-AM) and NADH show a broad absorbance band around 350 nm

  • Verify product formation through NMR spectroscopy as described in the literature

How is ALDH8A1 being investigated in cancer immunology research?

Recent multi-omics studies have revealed interesting connections between ALDH8A1 and cancer immunology:

• Tumor microenvironment correlations:

  • The ALDH gene signature (including ALDH8A1) correlates with immunosuppressive cell infiltration

  • High-risk ALDH signature tumors show elevated infiltration of immunosuppressive cell types including Tregs and macrophages

• Immune checkpoint associations:

  • ALDH risk score positively correlates with:

    • T cell co-inhibition (R = 0.26, p = 3.4×10⁻⁷)

    • Antigen-presenting cell co-inhibition (R = 0.29, p = 2.91×10⁻⁸)

    • Checkpoint expression (R = 0.31, p = 2.03×10⁻⁹)

  • Negative correlation with Type II interferon response (R = -0.38, p = 6.99×10⁻¹⁴)

• Research applications:

  • ALDH8A1 antibodies can be used to validate expression in tumor samples

  • Correlation studies between ALDH8A1 expression and infiltrating immune cell populations

  • Investigation of ALDH8A1 as a potential therapeutic target in combination with immunotherapy

What are the key considerations when using ALDH8A1 antibodies in single-cell analysis techniques?

For successful single-cell applications with ALDH8A1 antibodies:

• Compatibility with single-cell protocols:

  • Ensure antibody performance in dilute solutions required for single-cell techniques

  • Validate specificity at the single-cell level using appropriate controls

  • Consider using conjugated antibodies optimized for flow cytometry or mass cytometry

• Analytical approaches:

  • Correlate ALDH8A1 expression with cell population clustering as demonstrated in recent single-cell transcriptomics studies of HCC

  • Consider the 16 major cell populations identified in HCC single-cell studies when designing panels

  • Include markers for key cell types (e.g., M1/M2 macrophages, CD4+/CD8+ T cells, Tregs)

• Data interpretation:

  • Account for technical variability in single-cell protein measurements

  • Correlate protein expression with transcriptomic data when available

  • Consider ALDH8A1 expression in the context of cellular differentiation states