ALDH3A2 Antibody

What is ALDH3A2 and what cellular functions does it perform?

ALDH3A2, also known as Fatty Aldehyde Dehydrogenase (FALDH), is a key enzyme that plays a crucial role in the detoxification of harmful aldehydes, particularly alcohol-derived acetaldehyde . It catalyzes the NAD+-dependent oxidation of long-chain aliphatic aldehydes into fatty acids, which is vital for maintaining cellular health and lipid metabolism . This enzymatic activity prevents the accumulation of toxic aldehydes that can lead to cellular damage . ALDH3A2 is a 485 amino acid single-pass membrane protein predominantly localized to the cytoplasmic side of the endoplasmic reticulum . It is expressed across various tissues including the liver, heart, lung, brain, kidney, and placenta, indicating its broad physiological importance .

What types of ALDH3A2 antibodies are available for research applications?

Researchers have access to several types of ALDH3A2 antibodies suitable for different experimental applications. Monoclonal antibodies such as the G-9 mouse monoclonal IgG1 kappa light chain antibody can detect ALDH3A2 protein of human origin through western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . These antibodies are available in both non-conjugated forms and various conjugated formats including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor conjugates that expand their utility across different detection systems . Polyclonal antibodies, such as rabbit polyclonal IgG antibodies raised against synthetic peptides near the carboxy terminus of human ALDH3A2, offer complementary research tools with potentially broader epitope recognition .

How do ALDH3A2 isoforms differ and which isoforms can be detected with available antibodies?

At least four isoforms of ALDH3A2 arise from alternative splicing events, which may contribute to diverse functional roles in different tissues . The observed molecular weight of ALDH3A2 in experimental settings is approximately 68 kDa, while the calculated molecular weight is 54.8 kDa, suggesting post-translational modifications . When selecting an antibody, researchers should consider which isoforms they aim to detect. Most commercial antibodies are designed to recognize multiple ALDH3A2 isoforms but may have different specificities. For instance, some antibodies are specifically engineered to have no cross-reactivity to the related protein ALDH3A1, allowing for isoform-specific detection . Researchers should review the antibody documentation to confirm recognition of their target isoform and validate this specificity in their experimental system.

What are the optimal protocols for using ALDH3A2 antibodies in Western blotting?

For Western blotting applications with ALDH3A2 antibodies, researchers should follow these methodological considerations for optimal results:

• Sample Preparation: Extract proteins from tissues or cells using a buffer containing protease inhibitors to prevent ALDH3A2 degradation. For membrane-associated ALDH3A2, inclusion of detergents such as Triton X-100 or NP-40 is critical for solubilization.

• Protein Loading and Separation: Load 20-50 μg of total protein per lane. Given ALDH3A2's calculated molecular weight of 54.8 kDa (observed at approximately 68 kDa), use a 10-12% SDS-PAGE gel for optimal resolution .

• Transfer and Blocking: After electrophoresis, transfer proteins to PVDF or nitrocellulose membranes using standard methods. Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute ALDH3A2 primary antibodies (typically 1:500-1:2000, depending on the specific antibody) in blocking buffer and incubate overnight at 4°C.

• Detection: Use appropriate secondary antibodies conjugated to HRP or fluorescent tags, followed by standard detection methods. For enhanced sensitivity, consider using antibody bundles that include signal boosters, such as the m-IgG Fc BP-HRP or m-IgGκ BP-HRP bundles .

• Controls: Include positive controls from tissues known to express ALDH3A2 (liver, brain, kidney) and negative controls through RNA interference or samples from tissues with low ALDH3A2 expression.

How should immunohistochemistry with ALDH3A2 antibodies be optimized for tissue sections?

For immunohistochemistry (IHC) applications with ALDH3A2 antibodies, follow this methodological approach:

• Tissue Preparation: Mount 10 μm-thick sections of paraffin-embedded tissue on glass slides, deparaffinize in xylene, and rehydrate through a graded alcohol series .

• Antigen Retrieval: Perform high-temperature antigen retrieval using a water bath with an appropriate buffer (typically citrate buffer pH 6.0 or EDTA buffer pH 9.0) to expose epitopes that may be masked during fixation .

• Blocking Endogenous Activity: Quench endogenous peroxidases using 3% H₂O₂, followed by blocking with 5% BSA for 45 minutes at room temperature to reduce non-specific binding .

• Antibody Incubation: Incubate sections with ALDH3A2 antibody at an optimized dilution (typically 1:350 for commercial antibodies) overnight at 4°C .

• Detection System: Apply an appropriate secondary antibody for 60 minutes at room temperature, followed by visualization using a detection system such as the Dako EnVision System .

• Quantification: Use a semi-quantitative integration method for scoring, considering both the proportion of positively stained cells (1 = 0-10%, 2 = 10-25%, 3 = 50-75%, 4 = 75-100%) and staining intensity (0 = none, 1 = weak, 2 = moderate, 3 = strong) . Calculate the final IHC score by multiplying these values, with scores >6 considered high expression and ≤6 considered low expression .

What considerations are important when using ALDH3A2 antibodies for immunofluorescence studies?

For immunofluorescence (IF) studies with ALDH3A2 antibodies, researchers should consider:

• Cell Preparation: For cultured cells, grow on coverslips or chamber slides, fix with 4% paraformaldehyde, and permeabilize with 0.1-0.5% Triton X-100 to allow antibody access to intracellular ALDH3A2.

• Antibody Selection: Choose ALDH3A2 antibodies validated for IF applications. Consider directly conjugated antibodies (with fluorophores like FITC or Alexa Fluor) for single-step detection or unconjugated primary antibodies with fluorophore-labeled secondary antibodies for signal amplification .

• Co-localization Studies: Given ALDH3A2's predominant localization to the endoplasmic reticulum, consider co-staining with ER markers to confirm proper subcellular localization and antibody specificity.

• Controls: Include appropriate controls including primary antibody omission, isotype controls, and verification of specificity through RNA interference (such as with siRNA targeting sequences like 5-GCATTGCACCCGACTATAT-3) .

• Imaging: Use confocal microscopy for precise subcellular localization studies, particularly when examining ALDH3A2's association with the endoplasmic reticulum or other cellular compartments.

How can ALDH3A2 antibodies be applied in studying Sjögren-Larsson Syndrome?

Sjögren-Larsson Syndrome (SLS) is a serious autosomal recessive neurocutaneous disorder characterized by severe mental retardation, seizures, and speech defects that results from mutations in the gene encoding ALDH3A2 . When investigating SLS using ALDH3A2 antibodies, researchers should:

• Expression Analysis: Compare ALDH3A2 protein levels in patient-derived samples (skin fibroblasts, lymphoblasts) versus controls using quantitative Western blotting with ALDH3A2 antibodies to assess protein expression levels and potential truncated products.

• Subcellular Localization: Employ immunofluorescence with ALDH3A2 antibodies to examine potential mislocalization of mutant ALDH3A2 proteins, as some mutations may affect trafficking to the endoplasmic reticulum.

• Functional Studies: Combine antibody-based detection with enzyme activity assays to correlate ALDH3A2 protein levels with functional deficits in aldehyde metabolism in patient samples.

• Therapeutic Research: Use ALDH3A2 antibodies to monitor the restoration of protein expression in experimental therapeutic approaches such as gene therapy or pharmacological chaperones designed to correct misfolded ALDH3A2 proteins.

• Patient Stratification: Develop ALDH3A2 antibody-based assays that can distinguish between different types of mutations (null versus missense) to potentially correlate protein expression patterns with disease severity and progression.

What role can ALDH3A2 antibodies play in cancer research, particularly in relation to gastric cancer?

ALDH3A2 has emerged as a potential biomarker in gastric cancer (GC), with clinical implications for prognosis and treatment . Researchers investigating ALDH3A2 in cancer contexts should consider:

How can ALDH3A2 antibodies be integrated into studies of metabolic pathways?

Gene Set Enrichment Analysis (GSEA) has revealed that high ALDH3A2 expression is associated with enrichment of several metabolic pathways, including β-alanine metabolism, butanoate metabolism, fatty acid metabolism, propanoate metabolism, and valine leucine and isoleucine degradation . To study these metabolic connections:

• Pathway Analysis: Use ALDH3A2 antibodies in combination with antibodies against other enzymes in these metabolic pathways to perform co-immunoprecipitation experiments that may reveal protein-protein interactions or multienzyme complexes.

• Metabolic Flux Studies: Combine ALDH3A2 antibody-based protein quantification with metabolomics approaches to correlate enzyme levels with metabolite profiles in cells or tissues with varying ALDH3A2 expression.

• Regulatory Investigations: Employ chromatin immunoprecipitation (ChIP) techniques to identify transcription factors that may coordinately regulate ALDH3A2 and other metabolic enzymes, linking ALDH3A2 expression to broader metabolic programs.

• Subcellular Mapping: Use immunofluorescence with ALDH3A2 antibodies and markers for metabolic organelles to investigate the spatial organization of ALDH3A2-dependent metabolic pathways within cells.

What are common issues with ALDH3A2 antibodies in Western blotting and how can they be resolved?

Researchers may encounter several challenges when using ALDH3A2 antibodies for Western blotting:

• Multiple Bands: The presence of multiple isoforms (at least four) of ALDH3A2 can result in multiple bands . Distinguish true isoforms from non-specific binding by including appropriate controls (siRNA knockdown) and consulting antibody documentation for expected band patterns.

• Size Discrepancy: The observed molecular weight of ALDH3A2 (approximately 68 kDa) differs from the calculated molecular weight (54.8 kDa) . This discrepancy may reflect post-translational modifications such as glycosylation or phosphorylation. If unexpected band sizes appear, verify with alternative antibodies targeting different epitopes of ALDH3A2.

• Weak Signal: ALDH3A2's localization to the endoplasmic reticulum membrane may result in poor extraction and weak signals. Improve extraction by using buffers containing appropriate detergents (e.g., 1% Triton X-100) and consider signal enhancement systems like the m-IgG Fc BP-HRP or m-IgGκ BP-HRP bundles .

• Non-specific Binding: Some antibodies may exhibit cross-reactivity with related proteins. Select antibodies specifically engineered to have no cross-reactivity to related proteins like ALDH3A1 and include appropriate blocking steps (5% BSA or milk in TBST) to minimize background.

How should researchers interpret conflicting results between different ALDH3A2 antibodies?

When faced with discrepancies between results obtained using different ALDH3A2 antibodies:

• Epitope Differences: Consider that antibodies targeting different epitopes may yield different results, especially if certain epitopes are masked by protein interactions or post-translational modifications. Compare the immunogen information of each antibody to understand potential epitope differences.

• Validation Approach: Implement a multi-validation strategy using techniques like RNA interference (siRNA targeting ALDH3A2) to confirm antibody specificity . Observe if the signal decreases proportionally to the reduction in ALDH3A2 expression.

• Isoform Specificity: Determine if the different antibodies recognize distinct ALDH3A2 isoforms. Some antibodies may be pan-isoform while others may be isoform-specific, explaining disparate results across tissues with different isoform expression patterns.

• Technical Parameters: Evaluate differences in experimental conditions (fixation methods, antigen retrieval, detection systems) that might affect epitope accessibility and antibody binding. Standardize protocols when comparing antibodies to minimize technical variables.

• Cross-validation: Use orthogonal methods such as mass spectrometry or mRNA quantification (qPCR) to independently verify ALDH3A2 expression levels and resolve antibody-based discrepancies .

What considerations are important when quantifying ALDH3A2 expression in tissue samples?

When quantifying ALDH3A2 expression in tissue samples using antibody-based methods:

• Scoring System Development: Implement a robust semi-quantitative scoring system for IHC that accounts for both staining intensity and proportion of positive cells, such as the system used in gastric cancer research (intensity: 0-3, proportion: 1-4, final score: multiplication of both values) .

• Standardization: Establish consistent staining conditions, image acquisition parameters, and scoring criteria to enable reliable comparison across samples and studies.

• Pathologist Blinding: Have multiple pathologists who are blinded to the clinical data independently score the samples to eliminate bias, as practiced in published research .

• Reference Standards: Include known positive (liver, heart) and negative control tissues in each batch to calibrate scoring and account for staining variability.

• Cut-off Determination: Define clinically relevant cut-off values for "high" versus "low" expression through statistical approaches rather than arbitrary thresholds. For instance, scores greater than six might be considered high expression based on outcome correlation .

• Digital Pathology: Consider implementing digital image analysis for more objective quantification of staining intensity and positive cell proportion, reducing inter-observer variability.

How can ALDH3A2 antibodies contribute to immunotherapy research in cancer?

Recent findings suggest potential connections between ALDH3A2 and cancer immunotherapy:

• Immune Checkpoint Correlation: Research has demonstrated that ALDH3A2 expression negatively correlates with immune checkpoint molecules PDCD1, PDCD1LG2, and CTLA-4 . ALDH3A2 antibodies can be used to stratify tumors based on ALDH3A2 expression and predict potential responsiveness to checkpoint inhibitor therapies.

• Biomarker Development: Develop multiplexed immunohistochemistry panels that include ALDH3A2 antibodies alongside immune cell markers and checkpoint molecules to characterize the tumor immune microenvironment comprehensively.

• Functional Studies: Employ ALDH3A2 antibodies in mechanistic studies investigating how ALDH3A2 expression might modulate tumor-immune interactions, potentially through aldehyde metabolism affecting immune cell function or recruitment.

• Therapeutic Monitoring: Use ALDH3A2 antibodies to monitor changes in expression during immunotherapy treatment, potentially identifying adaptive resistance mechanisms or predictive response patterns.

• Combination Approaches: Investigate whether targeting ALDH3A2 (monitored via antibody-based detection) could enhance immunotherapy efficacy by altering metabolic properties of cancer cells or their microenvironment.

What new methodologies are emerging for ALDH3A2 protein analysis beyond traditional antibody applications?

Beyond conventional antibody applications, several emerging methodologies are enhancing ALDH3A2 protein analysis:

• Proximity Ligation Assays (PLA): This technique can detect protein-protein interactions involving ALDH3A2 with higher sensitivity than co-immunoprecipitation, potentially revealing new interaction partners in metabolic pathways.

• Mass Cytometry (CyTOF): By conjugating ALDH3A2 antibodies to metal isotopes, researchers can perform highly multiplexed single-cell analysis of ALDH3A2 alongside dozens of other proteins in heterogeneous samples.

• Super-resolution Microscopy: These techniques overcome the diffraction limit of conventional microscopy, allowing precise subcellular localization of ALDH3A2 within the endoplasmic reticulum and potential co-localization with functional partners.

• Spatial Transcriptomics: Combining ALDH3A2 antibody-based protein detection with spatially resolved transcriptomics can provide insights into the relationship between ALDH3A2 protein expression and local transcriptional programs within tissue microenvironments.

• Protein Turnover Analysis: Using pulsed stable isotope labeling with amino acids in cell culture (pSILAC) combined with ALDH3A2 immunoprecipitation can provide insights into the synthesis and degradation rates of ALDH3A2 under various physiological conditions.

Table 1: ALDH3A2 Antibody Selection Guide for Different Applications