ALDH5A1 Antibody

What is ALDH5A1 and why is it significant in neuroscience research?

ALDH5A1, also known as succinate semialdehyde dehydrogenase (SSADH), is a mitochondrial NAD(+)-dependent enzyme that plays a crucial role in the metabolism of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system. This enzyme catalyzes the conversion of succinate semialdehyde to succinate, which represents a key step in the degradation pathway of GABA .

ALDH5A1 is particularly significant in neuroscience research because deficiency in this enzyme leads to 4-hydroxybutyricaciduria (also called SSADH deficiency), a rare autosomal recessive disorder characterized by the accumulation of γ-hydroxybutyric acid (GHB) and other potentially toxic metabolites . This condition manifests with neurological symptoms including developmental delay, intellectual disability, ataxia, and epilepsy, with the severity of epilepsy typically increasing with age . Understanding ALDH5A1 function is essential for investigating the pathophysiology of this disorder and for broader studies of GABA metabolism in the brain.

What applications are ALDH5A1 antibodies commonly used for?

ALDH5A1 antibodies are versatile research tools employed across multiple experimental techniques:

• Western Blotting (WB): For detecting and quantifying ALDH5A1 protein in tissue or cell lysates, with typical dilutions ranging from 1:5000 to 1:50000

• Immunohistochemistry (IHC): For visualizing the distribution of ALDH5A1 in tissue sections, with recommended dilutions of 1:500 to 1:2000

• Immunofluorescence (IF): For cellular localization studies with dilutions of 1:200 to 1:800

• Flow Cytometry (FC): For quantifying ALDH5A1 in cell populations, typically using 0.40 μg per 10^6 cells in a 100 μl suspension

• Immunoprecipitation (IP): For isolating ALDH5A1 and its interaction partners

• ELISA: For quantitative measurement of ALDH5A1 in solution

When selecting an application, researchers should consider the specific experimental question and the most appropriate technique for obtaining reliable and reproducible results.

How do I select the appropriate ALDH5A1 antibody for my experiment?

Selecting the appropriate ALDH5A1 antibody requires consideration of several key factors:

• Host Species and Antibody Type: Available options include mouse monoclonal (IgG1 κ) antibodies that offer high specificity and consistency , and rabbit recombinant monoclonal antibodies that provide excellent reproducibility .

• Specific Applications: Verify that the antibody has been validated for your intended application. For example, if performing Western blotting, check if the antibody has demonstrated clear bands at the expected molecular weight (approximately 51-57 kDa for ALDH5A1) .

• Species Reactivity: Confirm that the antibody recognizes ALDH5A1 from your species of interest. Many ALDH5A1 antibodies react with human, mouse, and rat samples, but cross-reactivity should be verified .

• Conjugation: Determine whether a conjugated or unconjugated antibody is required. Options include unconjugated antibodies and those conjugated to HRP, PE, FITC, or various Alexa Fluor® dyes for direct detection .

• Epitope Recognition: Consider the specific region of ALDH5A1 that the antibody recognizes, especially if studying truncated forms or if certain domains are masked in your experimental system.

Review validation data and published literature using the antibody to ensure its reliability for your specific research context.

What is the expected molecular weight for ALDH5A1 detection?

ALDH5A1 is expected to be detected at approximately 51-57 kDa on Western blots, with slight variations depending on the specific antibody and sample preparation protocol. The calculated molecular weight based on the amino acid sequence (535 amino acids) is 57 kDa, while the observed molecular weight in many experimental systems is approximately 51 kDa .

This discrepancy between calculated and observed weights is not uncommon and could be attributed to several factors including post-translational modifications, protein folding, or proteolytic processing. When analyzing Western blot results, it is important to compare your observed band with positive controls and to verify specificity through appropriate negative controls.

What are the recommended dilutions and protocols for different applications?

The following table summarizes recommended dilutions for various applications of ALDH5A1 antibodies:

These dilutions should be optimized for each experimental system, as factors such as protein expression levels, sample preparation, and detection methods can affect optimal antibody concentration.

How do I optimize Western blot protocols for ALDH5A1 detection?

For optimal detection of ALDH5A1 by Western blot, consider the following methodological recommendations:

• Sample Preparation:

  • Use appropriate lysis buffers containing protease inhibitors to prevent degradation

  • Include mitochondrial extraction protocols for enhanced detection, as ALDH5A1 is a mitochondrial protein

• Gel Selection and Transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution around the 51-57 kDa range

  • Transfer proteins to nitrocellulose or PVDF membranes using standard protocols

• Blocking and Antibody Incubation:

  • Block membranes in 5% milk in TBS-0.1% Tween® 20

  • Incubate with primary ALDH5A1 antibody (1:5000-1:50000 dilution) overnight at 4°C

  • Wash thoroughly (four times in TBS-T) before secondary antibody incubation

• Detection and Controls:

  • Use HRP-conjugated secondary antibodies or direct HRP-conjugated primary antibodies for chemiluminescent detection

  • Include a loading control such as anti-Calnexin (CANX) antibody

  • For multiplexing, consider secondary antibodies with different fluorescent tags (e.g., 680RD)

• Troubleshooting:

  • If signal is weak, increase antibody concentration or extend incubation time

  • If background is high, increase washing steps or adjust blocking conditions

  • For multiple bands, verify specificity with knockout or knockdown controls

Following these optimization steps will enhance the specificity and sensitivity of ALDH5A1 detection in Western blot experiments.

How can I validate the specificity of ALDH5A1 antibodies?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For ALDH5A1 antibodies, consider implementing these validation approaches:

• Genetic Validation:

  • Test antibody on samples from ALDH5A1 knockout models or CRISPR-edited cell lines

  • Use siRNA or shRNA knockdown of ALDH5A1 to confirm signal reduction

• Recombinant Protein Controls:

  • Perform Western blot with purified recombinant ALDH5A1 protein as a positive control

  • Pre-adsorption test by incubating antibody with excess recombinant ALDH5A1 before application

• Multiple Antibody Validation:

  • Compare results using different antibodies targeting distinct epitopes of ALDH5A1

  • Verify consistent localization patterns across antibodies in immunostaining experiments

• Expression Pattern Analysis:

  • Confirm detection in tissues known to express ALDH5A1 (liver, heart, lung, brain, kidney, placenta)

  • Verify subcellular localization consistent with ALDH5A1's mitochondrial distribution

• Mass Spectrometry Confirmation:

  • Perform immunoprecipitation followed by mass spectrometry to confirm identity of the captured protein

Implementing multiple validation strategies provides the most robust confirmation of antibody specificity and increases confidence in experimental findings.

How can ALDH5A1 antibodies be used to study SSADH deficiency mechanisms?

ALDH5A1 antibodies serve as valuable tools for investigating the molecular mechanisms underlying SSADH deficiency through several sophisticated approaches:

• Protein Expression Analysis in Patient Samples:

  • Western blot and immunohistochemistry can quantify ALDH5A1 protein levels in patient-derived fibroblasts, lymphoblasts, or postmortem tissue samples

  • This allows correlation between specific mutations and residual protein expression

• Functional Studies of Variant Proteins:

  • Site-directed mutagenesis can be used to introduce patient-specific mutations into ALDH5A1 expression constructs

  • Transfection into HEK293 cells followed by Western blot analysis can determine if mutations affect protein stability or expression levels

  • Activity assays combined with immunoblotting can correlate protein expression with enzymatic function

• Cellular Consequence Investigation:

  • Immunofluorescence microscopy using ALDH5A1 antibodies can assess subcellular localization of mutant proteins

  • Co-staining with mitochondrial markers can determine if mutations disrupt proper targeting to mitochondria

• Therapeutic Development:

  • ALDH5A1 antibodies can monitor protein expression in cells treated with compounds designed to rescue mutant protein function

  • Measure effects of chaperone therapies or gene therapy approaches on protein expression and localization

What are the considerations for using ALDH5A1 antibodies in studies of GABA metabolism?

When employing ALDH5A1 antibodies to investigate GABA metabolism, researchers should consider these key methodological aspects:

• Integrated Pathway Analysis:

  • Use multiplexing approaches that combine ALDH5A1 antibodies with antibodies against other GABA metabolic enzymes (GAD, GABA-T) to visualize the entire pathway

  • Co-immunoprecipitation experiments can identify protein-protein interactions within the GABA metabolic complex

• Regional Brain Expression Patterns:

  • Employ immunohistochemistry with ALDH5A1 antibodies to map expression across different brain regions

  • Correlate expression patterns with regional GABA concentrations and neurophysiological functions

• Cell-Type Specific Analysis:

  • Combine ALDH5A1 immunostaining with neuronal, astrocytic, or oligodendrocytic markers to determine cell-type specific expression

  • This approach is crucial as GABA metabolism involves intercellular metabolic coupling

• Activity-Dependent Regulation:

  • Investigate changes in ALDH5A1 expression or localization in response to neuronal activity

  • Establish correlations between enzyme levels and GABA/GHB concentrations in various physiological states

• Developmental Studies:

  • Use ALDH5A1 antibodies to track developmental changes in enzyme expression

  • Correlate with the maturation of GABAergic neurotransmission and inhibitory circuit development

These approaches enable a comprehensive understanding of how ALDH5A1 functions within the complex network of GABA metabolism and how its dysfunction contributes to neurological disorders.

How can ALDH5A1 antibodies be employed to investigate genotype-phenotype relationships in SSADH deficiency?

Investigating genotype-phenotype relationships in SSADH deficiency using ALDH5A1 antibodies involves several sophisticated methodological approaches:

• Mutation-Specific Protein Analysis:

  • Western blot with ALDH5A1 antibodies can quantify residual protein levels associated with specific mutations

  • Compare expression levels across patients with different mutations and correlate with clinical severity scores (CSS)

• Structure-Function Correlation:

  • Immunoprecipitation followed by activity assays can determine how different mutations affect both protein expression and enzymatic function

  • This approach has revealed that some variants like c.1226G>A (p.Gly409Asp) may correlate with severe clinical presentations

• Protein Trafficking Studies:

  • Immunofluorescence microscopy can assess whether specific mutations affect subcellular localization

  • Compare mutations that cause protein misfolding versus those that primarily affect catalytic activity

• Age-Dependent Expression Patterns:

  • Analyze ALDH5A1 expression in relation to age-dependent symptoms, such as the increased prevalence of epilepsy and obsessive-compulsive disorder in older patients

  • This approach can help elucidate why certain genotypes show variable phenotypic expression with age

• Risk Stratification for SUDEP:

  • Study specific variants associated with sudden unexpected death in epilepsy (SUDEP) using antibody-based protein characterization

  • Variants such as c.1015-2A>C, c.1005C>A (p.Asn335Lys), and c.1226G>A (p.Gly409Asp) have been identified in SUDEP cases

Research has shown that while direct genotype-phenotype correlations are not consistently observed across all patients, specific variants may predict particular clinical outcomes . For example, certain missense variants have been associated with milder phenotypes (normal IQ), while others consistently correlate with more severe presentations. This nuanced approach using ALDH5A1 antibodies helps refine our understanding of how specific molecular alterations translate to clinical manifestations.

What techniques can be used to study post-translational modifications of ALDH5A1?

Investigating post-translational modifications (PTMs) of ALDH5A1 requires specialized antibody-based approaches:

• Modification-Specific Antibodies:

  • Use antibodies that specifically recognize phosphorylated, acetylated, or ubiquitinated forms of ALDH5A1

  • Employ these in Western blot or immunoprecipitation to quantify modified fractions of the protein

• Two-Dimensional Gel Electrophoresis:

  • Combine with ALDH5A1 antibody detection to identify charge variants resulting from phosphorylation or other PTMs

  • This approach can separate multiple modified forms of the protein

• Immunoprecipitation-Mass Spectrometry Workflow:

  • Immunoprecipitate ALDH5A1 using validated antibodies

  • Analyze the purified protein by mass spectrometry to identify and map specific PTM sites

  • This technique can reveal previously uncharacterized modifications

• Sequential Immunoprecipitation:

  • First immunoprecipitate with ALDH5A1 antibody, then probe with modification-specific antibodies

  • Alternatively, immunoprecipitate with modification-specific antibodies and probe with ALDH5A1 antibody

• Proximity Ligation Assays:

  • Combine ALDH5A1 antibodies with antibodies against enzymes that catalyze PTMs

  • This approach can reveal spatial relationships between ALDH5A1 and PTM-regulating enzymes

Understanding PTMs of ALDH5A1 is particularly relevant as they may influence enzyme activity, protein stability, or subcellular localization, potentially contributing to pathophysiological mechanisms in conditions like SSADH deficiency where seemingly minor alterations in enzyme function can have profound neurological consequences.

How do I troubleshoot weak or absent signals when using ALDH5A1 antibodies?

When encountering weak or absent signals with ALDH5A1 antibodies, implement this systematic troubleshooting approach:

• Sample Preparation Issues:

  • Ensure complete cell/tissue lysis, particularly of mitochondrial fractions where ALDH5A1 is localized

  • Verify protein integrity by checking other mitochondrial proteins

  • Include fresh protease inhibitors to prevent degradation

• Antibody-Related Factors:

  • Check antibody expiration date and storage conditions

  • Titrate antibody concentration (try higher concentrations, e.g., 1:1000 instead of 1:5000 for Western blot)

  • Consider switching to a different antibody clone or one recognizing a different epitope

• Protocol Optimization:

  • For Western blotting: extend primary antibody incubation time (overnight at 4°C), ensure proper transfer of proteins in the 51-57 kDa range

  • For IHC/IF: optimize antigen retrieval (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • For flow cytometry: verify permeabilization efficiency for intracellular staining

• Detection System Issues:

  • Ensure secondary antibody compatibility with primary antibody host species

  • Prepare fresh detection reagents (ECL, DAB, etc.)

  • Extend exposure time for Western blots or increase detector sensitivity

• Positive Controls:

  • Include tissues known to express high levels of ALDH5A1 (liver, brain)

  • Use recombinant ALDH5A1 protein as a positive control

Systematically addressing these factors will help identify the source of the problem and achieve successful ALDH5A1 detection.

What controls should be included when working with ALDH5A1 antibodies?

Robust experimental design for ALDH5A1 antibody applications should include these essential controls:

• Positive Controls:

  • Tissues or cell lines with known ALDH5A1 expression (L02 cells, HepG2 cells, brain tissue)

  • Recombinant ALDH5A1 protein or overexpression systems

  • For Western blot, include a loading control such as Calnexin (CANX)

• Negative Controls:

  • ALDH5A1 knockout or knockdown samples when available

  • Isotype control antibodies matching the ALDH5A1 antibody class (e.g., mouse IgG1 κ)

  • Primary antibody omission to assess secondary antibody specificity

• Specificity Controls:

  • Peptide competition assay: pre-incubating the antibody with excess immunizing peptide should abolish specific signal

  • Multiple antibodies recognizing different epitopes should show similar patterns

• Technical Controls:

  • For IHC/IF: include autofluorescence or endogenous peroxidase controls

  • For flow cytometry: include unstained, single-stained, and FMO (fluorescence minus one) controls

  • For ELISA: include standard curves and blank wells

• Cross-Reactivity Assessment:

  • Test samples from multiple species if cross-reactivity is claimed

  • Include samples from closely related ALDH family members to verify specificity

Incorporating these controls ensures the validity of results and facilitates troubleshooting if unexpected findings occur.

How are ALDH5A1 antibodies used in epilepsy research?

ALDH5A1 antibodies play a crucial role in epilepsy research, particularly in understanding SSADH deficiency-related seizures:

• Age-Dependent Expression Analysis:

  • ALDH5A1 antibodies can track changes in protein expression with age, correlating with the increased prevalence of epilepsy in adolescent/adult SSADH deficiency patients (83% in patients >12 years vs. 11% in patients <12 years)

  • This approach helps elucidate mechanisms underlying the age-dependent emergence of seizures

• SUDEP Risk Assessment:

  • Specific ALDH5A1 variants have been associated with sudden unexpected death in epilepsy (SUDEP)

  • Antibody-based protein characterization of these variants (c.1015-2A>C, c.1005C>A, c.1226G>A, and c.608C>G) helps understand their functional consequences

• Therapeutic Target Validation:

  • ALDH5A1 antibodies can monitor protein expression levels in response to antiepileptic treatments

  • This approach helps validate ALDH5A1 as a therapeutic target and assess drug efficacy at the molecular level

• Network Excitability Studies:

  • Combining ALDH5A1 immunostaining with markers of neuronal activation helps correlate GABA metabolism alterations with network hyperexcitability

  • This multi-antibody approach provides insights into seizure generation mechanisms

• Model System Validation:

  • ALDH5A1 antibodies verify protein expression in animal models of SSADH deficiency

  • Comparing human patient samples with model systems confirms the translational relevance of experimental findings

These applications demonstrate how ALDH5A1 antibodies contribute to understanding the complex relationship between GABA metabolism, GHB accumulation, and epileptogenesis in both clinical settings and experimental models.

What considerations are important when using ALDH5A1 antibodies in patient-derived samples?

When applying ALDH5A1 antibodies to patient-derived samples, researchers should consider these specialized methodological aspects:

• Sample Variability and Standardization:

  • Patient samples show greater variability than cell lines; standardize collection, processing, and storage procedures

  • Include age-matched and sex-matched controls when analyzing patient tissues or cells

  • Document medication status as some treatments may affect ALDH5A1 expression

• Limited Sample Availability:

  • Optimize protocols for small sample volumes (micro-Western blots, single-cell techniques)

  • Consider multiplexing approaches to maximize data from limited material

  • Validate antibodies on similar sample types before precious patient samples are used

• Genetic Background Considerations:

  • Different ALDH5A1 variants may affect antibody binding differently

  • When studying compound heterozygotes (common in SSADH deficiency) , consider how each mutation might affect antibody recognition

  • Sequence verification of the epitope region is recommended for definitive interpretation

• Technical Adaptations:

  • For fibroblasts or lymphoblasts: optimize lysis conditions for these specific cell types

  • For brain tissue: consider postmortem changes and fixation effects on epitope accessibility

  • For blood samples: account for lower ALDH5A1 expression compared to brain or liver

• Ethical and Consent Considerations:

  • Ensure appropriate informed consent for immunological studies

  • Consider anonymous coding systems to protect patient identity

  • Follow institutional ethical guidelines for patient-derived materials

These considerations help ensure reliable and reproducible results when applying ALDH5A1 antibodies to the study of patient samples, particularly in rare disorders like SSADH deficiency.