ALDH1A3 Antibody

What is ALDH1A3 and what biological functions does it serve?

ALDH1A3 is a member of the aldehyde dehydrogenase family that catalyzes the NAD-dependent oxidation of aldehyde substrates to their corresponding carboxylic acids. It demonstrates high specificity for all-trans-retinal as a substrate, converting it to all-trans-retinoic acid, though it can also accept acetaldehyde as a substrate with lower affinity . This enzyme plays a critical role in the biosynthesis of normal levels of retinoate in embryonic ocular and nasal regions, making it essential for proper embryonic development of the eye and nasal structures . In cancer biology, ALDH1A3 is one of the primary contributors to high Aldefluor activity that defines ALDH+ cancer stem cells in various cancers including breast, melanoma, glioblastoma, lung, and prostate cancer . The enzyme regulates cancer-promoting processes and influences the abundance of distinct breast cancer stem cell populations, suggesting its importance in tumor progression and metastasis .

What types of ALDH1A3 antibodies are available for experimental applications?

ALDH1A3 antibodies are available in several formats to accommodate various experimental needs. The primary classifications include:

• Host Species: Rabbit polyclonal antibodies (such as ABIN5518802) and mouse monoclonal antibodies (like OTI4E8) are commonly available .

• Clonality: Both polyclonal antibodies, which recognize multiple epitopes on the antigen, and monoclonal antibodies, which bind to a single epitope, are available for ALDH1A3 detection .

• Binding Specificity: Different antibodies target specific amino acid regions of ALDH1A3, such as AA 37-154, AA 1-100, AA 24-51, AA 318-348, or AA 332-509, allowing researchers to target different functional domains of the protein .

• Conjugation Status: Most ALDH1A3 antibodies are available in unconjugated forms, but some may be conjugated to fluorophores or enzymes for direct detection .

The selection of the appropriate antibody depends on the specific experimental questions and techniques planned.

What applications are ALDH1A3 antibodies validated for?

ALDH1A3 antibodies have been validated for multiple research applications, with different antibodies showing suitability for specific techniques:

• Western Blotting (WB): Most ALDH1A3 antibodies are validated for protein detection via western blotting, allowing quantification of expression levels in tissue or cell lysates .

• Immunohistochemistry (IHC): Both paraffin-embedded (IHC-P) and frozen section protocols are supported by various ALDH1A3 antibodies, enabling localization studies in tissues .

• Flow Cytometry (FACS): Select antibodies like the mouse monoclonal (OTI4E8) are suitable for intracellular flow cytometry, facilitating identification and isolation of ALDH1A3-expressing cells .

• Immunoprecipitation (IP): Some antibodies are validated for immunoprecipitation, allowing researchers to study protein-protein interactions involving ALDH1A3 .

• Immunocytochemistry (ICC): Certain antibodies can be used for cellular localization studies in cultured cells .

When selecting an antibody, researchers should verify that it has been validated for their intended application in relevant species and cell types.

What species reactivity do available ALDH1A3 antibodies demonstrate?

The species reactivity of ALDH1A3 antibodies varies by product, but many show cross-reactivity across human, mouse, and rat samples. For example:

• The rabbit polyclonal antibody ABIN5518802 has been tested and validated for reactivity with human, mouse, and rat ALDH1A3 .

• The mouse monoclonal antibody OTI4E8 reacts with human and mouse samples .

This cross-reactivity is facilitated by the high sequence conservation of ALDH1A3 across species. Human ALDH1A3 shares 89% amino acid sequence identity with mouse ALDH1A3 and 87.3% with rat ALDH1A3 . When working with species not explicitly listed in validation studies, researchers should perform preliminary tests to confirm antibody reactivity before proceeding with full experiments.

How can ALDH1A3 antibodies be utilized to investigate cancer stem cell heterogeneity?

ALDH1A3 antibodies can be instrumental in exploring the complex relationship between different cancer stem cell populations, particularly in breast cancer. Research has demonstrated that ALDH1A3 regulates the balance between two distinct stem cell populations: ALDH+ cells and CD24-CD44+ cells . When designing experiments to investigate this heterogeneity:

• Dual marker flow cytometry: Combine ALDH1A3 antibody staining with CD24 and CD44 antibodies to simultaneously identify and quantify both stem cell populations. This approach allows for correlation analysis between ALDH1A3 expression levels and the prevalence of each stem cell phenotype .

• Knockdown/overexpression validation: Include ALDH1A3 knockdown (in high-expressing lines like MDA-MB-468 and HCC1806) or overexpression (in low-expressing lines like MDA-MB-231) experiments to establish causality in observed relationships. Flow cytometry analysis following these genetic manipulations can reveal how ALDH1A3 directly influences stem cell population dynamics .

• Transcriptional analysis: Complement protein detection with RT-qPCR analysis of CD24 and CD44 transcript levels to determine whether ALDH1A3 regulates these markers at the transcriptional level. Research has shown that ALDH1A3 knockdown significantly increases CD44 expression and decreases CD24 expression, while ALDH1A3 overexpression increases CD24 expression .

This multi-faceted approach can provide comprehensive insights into how ALDH1A3 serves as a molecular switch controlling stem cell population balance in cancer.

What experimental approaches can validate the specificity of ALDH1A3 antibodies?

Validating antibody specificity is crucial for reliable experimental results. For ALDH1A3 antibodies, consider these validation approaches:

• Genetic manipulation controls: Use cell lines with ALDH1A3 knockdown (via siRNA or CRISPR) or overexpression as positive and negative controls. Signal reduction after knockdown or enhancement after overexpression confirms specificity. The TNBC cell lines MDA-MB-468 and HCC1806 (high ALDH1A3 expressors) and MDA-MB-231 (low ALDH1A3 expressor) provide good model systems for such validation .

• Recombinant protein competition: Pre-incubate the antibody with purified recombinant ALDH1A3 protein (such as E.coli-derived human ALDH1A3 recombinant protein) before application to samples. Signal reduction indicates specific binding .

• Cross-reactivity assessment: Test against related ALDH family members, particularly ALDH1A1, which also contributes to Aldefluor activity. Antibodies like ABIN5518802 are reported to have no cross-reactivity with other proteins, but this should be verified for each experimental system .

• Multiple antibody comparison: Use antibodies targeting different epitopes of ALDH1A3 (such as AA 37-154, AA 24-51, or AA 318-348) and compare staining patterns. Consistent results across different antibodies increase confidence in specificity .

• Immunoprecipitation-mass spectrometry: For definitive validation, immunoprecipitate with the ALDH1A3 antibody and identify captured proteins by mass spectrometry to confirm selective enrichment of ALDH1A3.

How can researchers combine ALDH1A3 antibody detection with functional assays to investigate its role in retinoic acid signaling?

ALDH1A3 converts retinal to retinoic acid, which induces gene expression changes by binding to hormone receptor ligands . To investigate this signaling pathway:

• Sequential retinal treatment and antibody staining: Treat cells with retinal (the ALDH1A3 substrate) for 24 hours, then analyze changes in CD24-CD44+ populations through antibody staining. This approach reveals how substrate availability affects ALDH1A3-mediated signaling outcomes .

• Reporter assays with antibody validation: Implement retinoic acid response element (RARE) reporter assays alongside ALDH1A3 antibody staining to correlate enzyme levels with signaling output. Compare wild-type cells with ALDH1A3 knockdown cells to establish the specific contribution of ALDH1A3 to retinoic acid production.

• Gene expression analysis: Follow ALDH1A3 antibody-based cell sorting with RNA sequencing or targeted RT-qPCR of retinoic acid-responsive genes. This approach links ALDH1A3 protein levels directly to transcriptional outcomes of the signaling pathway.

• Functional rescue experiments: In ALDH1A3 knockdown cells, compare the effects of exogenous retinoic acid addition versus retinal addition. If retinoic acid but not retinal rescues the phenotype, this confirms that the observed effects depend on ALDH1A3's enzymatic conversion of retinal to retinoic acid.

These combined approaches provide mechanistic insights beyond what either antibody detection or functional assays could reveal independently.

What methodological approaches can elucidate the role of ALDH1A3 in cellular metabolism?

ALDH1A3 is implicated in metabolic processes beyond retinoic acid production. To investigate these functions:

• Glycolysis assessment: Use the Glycolysis Assay [Extracellular activation] to measure glycolytic activity in cells with modified ALDH1A3 expression. Seed 3 × 10^4 cells in black-walled plates, subject them to overnight CO2 purge, and measure fluorescence at Ex/Em = 380/615 nm at 30-second intervals for 1 hour. Normalize glycolytic activity to cell number for comparative analysis .

• ATP synthase activity measurement: Implement an absorbance microplate assay using 5 mg/mL protein isolated from cells with varied ALDH1A3 expression. After detergent treatment and enzyme immobilization, add phospholipid mix and measure absorbance at 340 nm at 1-minute intervals for 30 minutes at 30°C to calculate ATP synthase activity .

• Real-time ATP production analysis: Utilize the Seahorse XF Real-Time ATP Rate Assay to measure oxygen consumption rate (OCR), extracellular acidification rate (EACR), and ATP production from both oxidative phosphorylation and glycolysis. For optimal results, seed 25,000 cells per well for MDA-MB-468 and HCC1806 cells or 35,000 cells per well for MDA-MB-231 cells .

• Metabolic inhibitor studies: Combine ALDH1A3 antibody staining with metabolic inhibitor treatments (such as 2-deoxyglucose at 5 mM for 48 hours) to understand how ALDH1A3-expressing cells respond to metabolic stress conditions .

These approaches allow researchers to establish connections between ALDH1A3 expression and specific metabolic pathways that may contribute to cancer progression and therapeutic resistance.

What considerations are important when using ALDH1A3 antibodies to investigate its role in chemoresistance?

ALDH1A3 contributes to chemoresistance in multiple cancers, including melanoma, breast, prostate, glioblastoma, and colon cancer . When designing experiments to study this association:

• Sequential chemotherapy and antibody staining: Treat cancer cell lines with clinically relevant chemotherapeutic agents at various concentrations and timepoints, followed by ALDH1A3 antibody staining to assess whether treatment selectively enriches for ALDH1A3-expressing cells.

• Sorting-based functional assays: Use ALDH1A3 antibody-based cell sorting to separate high and low expressing populations, then compare their chemoresistance profiles using viability assays after drug treatment. This approach directly links ALDH1A3 expression levels to treatment response.

• Patient-derived xenograft (PDX) models: Apply ALDH1A3 antibody staining to track changes in ALDH1A3 expression in PDX models before and after chemotherapy treatment, correlating expression patterns with treatment response and recurrence.

• Multi-parameter analysis: Combine ALDH1A3 antibody staining with markers of apoptosis, cell cycle, and DNA damage to characterize the specific protective mechanisms conferred by ALDH1A3 expression.

• Longitudinal studies: In clinical samples, use ALDH1A3 antibodies to assess expression before treatment, during treatment, and at recurrence to establish temporal relationships between ALDH1A3 expression and therapeutic resistance.

These approaches can elucidate the mechanistic basis of ALDH1A3-mediated chemoresistance and potentially identify strategies to overcome this resistance.

What protocols yield optimal results for ALDH1A3 detection in various applications?

For reliable ALDH1A3 detection across different experimental techniques, consider these optimized protocols:

Western Blotting Protocol:

• Lyse cells or tissues in RIPA buffer supplemented with protease inhibitors.

• Separate 20-50 μg of protein by SDS-PAGE (10% gel).

• Transfer to PVDF membrane (wet transfer recommended for optimal results).

• Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Incubate with primary ALDH1A3 antibody (1:500-1:1000 dilution) overnight at 4°C.

• Wash 3x with TBST, 5 minutes each.

• Incubate with appropriate HRP-conjugated secondary antibody (1:2000-1:5000) for 1 hour at room temperature.

• Wash 3x with TBST, 5 minutes each.

• Develop using ECL substrate and image.

• Expected molecular weight: approximately 56 kDa .

Immunohistochemistry Protocol (Paraffin-embedded Sections):

• Deparaffinize and rehydrate sections.

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Block endogenous peroxidase with 3% H₂O₂ for 10 minutes.

• Block non-specific binding with 5% normal serum in PBS for 1 hour.

• Incubate with primary ALDH1A3 antibody (1:100-1:200 dilution) overnight at 4°C.

• Wash 3x with PBS, 5 minutes each.

• Apply appropriate biotinylated secondary antibody for 30 minutes at room temperature.

• Wash 3x with PBS, 5 minutes each.

• Apply avidin-biotin complex for 30 minutes.

• Develop with DAB substrate and counterstain with hematoxylin .

Flow Cytometry Protocol:

• Harvest cells and wash with PBS containing 2% FBS.

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes.

• Block with 2% BSA in PBS for 30 minutes.

• Incubate with primary ALDH1A3 antibody (1:100 dilution) for 1 hour at room temperature.

• Wash 3x with PBS containing 2% FBS.

• Incubate with fluorophore-conjugated secondary antibody for 30 minutes at room temperature.

• Wash 3x with PBS containing 2% FBS.

• Analyze by flow cytometry, using appropriate isotype controls to set gating parameters .

How should researchers troubleshoot common issues with ALDH1A3 antibody staining?

When encountering difficulties with ALDH1A3 antibody staining, consider these troubleshooting approaches:

Weak or No Signal:

• Antibody concentration: Titrate antibody concentrations systematically (typically 1:50 to 1:1000) to determine optimal dilution for your specific sample type.

• Epitope masking: Test different antigen retrieval methods (heat-induced with citrate buffer pH 6.0 versus EDTA buffer pH 9.0) to ensure proper epitope exposure.

• Expression levels: Confirm ALDH1A3 expression in your sample type using positive control tissues or cell lines with known high expression (e.g., MDA-MB-468 or HCC1806 cells) .

• Antibody viability: Verify antibody activity using a simple dot blot with recombinant ALDH1A3 protein.

• Detection system sensitivity: Switch to a more sensitive detection system (e.g., polymer-based instead of ABC method for IHC).

High Background:

• Blocking optimization: Increase blocking time or concentration (5-10% normal serum corresponding to secondary antibody host).

• Secondary antibody cross-reactivity: Pre-absorb secondary antibody with tissue powder from the species being examined.

• Endogenous enzyme activity: For IHC, ensure thorough quenching of endogenous peroxidase (3% H₂O₂ for 10-15 minutes) or phosphatase activity.

• Washing stringency: Increase washing steps (5-6 times, 5 minutes each) with PBS-T (0.1% Tween-20).

• Antibody specificity: Consider using a different ALDH1A3 antibody targeting a different epitope to verify results.

Inconsistent Staining:

• Sample preparation standardization: Standardize fixation times and conditions across all samples.

• Batch processing: Process all experimental samples in the same batch to minimize technical variations.

• Internal controls: Include known positive and negative controls in each experimental run.

• Automated systems: Consider using automated staining platforms to reduce operator-dependent variations.

What considerations are important when designing ALDH1A3 knockdown or overexpression validation experiments?

When modifying ALDH1A3 expression for experimental validation:

• Selection of appropriate cell models: Choose cell lines with relevant endogenous ALDH1A3 expression levels. MDA-MB-468 and HCC1806 cells have high ALDH1A3 expression (ideal for knockdown), while MDA-MB-231 cells have low endogenous expression (suitable for overexpression studies) .

• Knockdown verification: When using siRNA or shRNA approaches, validate knockdown efficiency through:

  • Western blotting with ALDH1A3 antibody to quantify protein reduction

  • RT-qPCR to confirm mRNA downregulation

  • Functional assays such as Aldefluor to verify reduced enzymatic activity

  Aim for at least 70-80% reduction in expression for meaningful functional analysis .

• Overexpression calibration: For overexpression models:

  • Use vectors with appropriate promoters (CMV for high expression, PGK for moderate expression)

  • Create stable cell lines for long-term studies to avoid transient expression variability

  • Verify expression levels via western blotting and RT-qPCR

  • Include enzymatically inactive mutants as controls to distinguish catalytic from non-catalytic functions

• Experimental timing: Consider the temporal dynamics of ALDH1A3 activity when designing experiments:

  • For siRNA knockdown: Optimal window is typically 48-72 hours post-transfection

  • For stable overexpression: Allow 2-3 passages for expression stabilization

  • For inducible systems: Determine appropriate induction timeframe through time-course experiments

• Downstream validation: Confirm that modification of ALDH1A3 affects expected downstream processes:

  • Measure retinoic acid production

  • Assess CD24/CD44 expression changes

  • Evaluate metabolic parameters (glycolysis, ATP production)

  • Test functional properties (proliferation, migration, chemoresistance)

This comprehensive validation approach ensures that observed phenotypes are specifically attributable to ALDH1A3 modulation.

How can ALDH1A3 antibodies be integrated into single-cell analysis workflows?

Single-cell technologies provide unprecedented resolution for analyzing cellular heterogeneity. To incorporate ALDH1A3 antibodies into these workflows:

• Single-cell protein profiling: Adapt ALDH1A3 antibody staining for mass cytometry (CyTOF) or spectral flow cytometry by using metal-conjugated or fluorochrome-conjugated antibodies. These platforms allow simultaneous detection of ALDH1A3 with dozens of other proteins to create comprehensive cellular phenotypes.

• Multimodal single-cell analysis: Combine ALDH1A3 antibody-based cell sorting with single-cell RNA sequencing to correlate protein expression with transcriptomic profiles. This approach can reveal how ALDH1A3 protein levels relate to global gene expression patterns in individual cells.

• Spatial transcriptomics integration: Use ALDH1A3 antibodies for immunofluorescence staining of tissue sections in conjunction with spatial transcriptomics methods. This approach preserves spatial context while providing molecular insights into ALDH1A3-expressing cells and their microenvironment.

• Live-cell imaging applications: Adapt cell-permeable ALDH1A3 antibody fragments for live-cell imaging to track ALDH1A3-expressing cells over time, potentially revealing dynamic changes in expression during processes like cell division, migration, or drug response.

• Microfluidic applications: Implement ALDH1A3 antibody staining in microfluidic single-cell analysis platforms to correlate ALDH1A3 expression with functional outputs such as secreted factors or cell-cell interaction behaviors.

These approaches can provide deeper insights into the heterogeneity and functional significance of ALDH1A3 expression in complex biological systems.

What are the considerations for using ALDH1A3 antibodies in translational research and clinical applications?

As ALDH1A3 research moves toward clinical applications, consider these translational aspects:

• Diagnostic biomarker development: When developing ALDH1A3 as a prognostic or predictive biomarker:

  • Establish standardized immunohistochemistry protocols with specific scoring criteria

  • Validate antibody performance across different tissue preservation methods

  • Determine threshold values that correlate with clinical outcomes

  • Account for intratumoral heterogeneity through multiple sampling

• Companion diagnostics: For therapies targeting ALDH1A3 or pathways it regulates:

  • Select antibodies with high specificity and sensitivity for the clinical setting

  • Develop protocols compatible with clinical laboratory workflows

  • Establish quality control metrics for reliable patient stratification

  • Consider automated image analysis for quantitative assessment

• Tissue microarray applications: When screening large cohorts:

  • Validate antibody performance on tissue microarrays versus whole sections

  • Account for sampling bias through appropriate core number and size

  • Implement multi-institutional standardization for consistent scoring

  • Correlate expression with comprehensive clinical metadata

• Circulating tumor cell (CTC) detection: For liquid biopsy applications:

  • Optimize antibody concentrations for rare cell detection

  • Implement dual-marker strategies (e.g., ALDH1A3 with epithelial markers)

  • Validate detection sensitivity and specificity with spiked-in controls

  • Correlate CTC ALDH1A3 expression with clinical outcomes

• Therapeutic response monitoring: For longitudinal assessment:

  • Establish baseline ALDH1A3 expression before treatment initiation

  • Develop minimally invasive sampling protocols for repeated assessment

  • Correlate expression changes with treatment response metrics

  • Consider combined assessment with other resistance markers

These considerations help bridge the gap between laboratory research and clinical application, maximizing the translational impact of ALDH1A3 antibody-based research.