ALDH7A1 Antibody

What is ALDH7A1 and why is it important in research?

ALDH7A1 is a multifunctional enzyme that mediates several important protective effects in cells. It metabolizes betaine aldehyde to betaine, which serves as an important cellular osmolyte and methyl donor. This enzyme also protects cells from oxidative stress by metabolizing various lipid peroxidation-derived aldehydes. Additionally, ALDH7A1 plays a crucial role in lysine catabolism . Also known as alpha-aminoadipic semialdehyde dehydrogenase, antiquitin-1, or betaine aldehyde dehydrogenase, this protein is involved in multiple biological pathways that make it relevant for research in neurological disorders, stress response, and metabolic regulation .

What applications are ALDH7A1 antibodies suitable for?

ALDH7A1 antibodies are validated for multiple experimental applications:

The selection of application should be guided by experimental goals, with particular attention to antibody validation for specific model systems .

What species reactivity can be expected from ALDH7A1 antibodies?

Most commercially available ALDH7A1 antibodies demonstrate cross-reactivity with multiple species, which is beneficial for comparative studies. Based on validation data, the following species reactivities have been confirmed:

• Human: Well-established reactivity across multiple antibody clones

• Mouse: Confirmed for many antibodies, particularly useful for animal models

• Rat: Validated for select antibodies

When working with other species, preliminary validation is recommended as reactivity may vary based on epitope conservation across species .

What are the recommended storage conditions for ALDH7A1 antibodies?

For optimal antibody performance and longevity, ALDH7A1 antibodies should be stored according to the following guidelines:

• Temperature: -20°C for long-term storage; some formulations require -80°C

• Avoid freeze/thaw cycles: Aliquot antibodies before freezing to minimize repeated freeze/thaw cycles

• Buffer composition: Typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Precautions: Note that most preparations contain sodium azide, which should be handled according to laboratory safety protocols as it is hazardous

Following these storage recommendations will help maintain antibody binding capacity and specificity over time.

How should I select between polyclonal and monoclonal ALDH7A1 antibodies for my research?

The choice between polyclonal and monoclonal ALDH7A1 antibodies depends on your experimental requirements:

Polyclonal Antibodies (e.g., rabbit polyclonal ab236663):

• Advantages: Recognize multiple epitopes, potentially higher sensitivity for low-abundance targets, useful when protein conformation might be altered

• Recommended for: Initial protein detection studies, applications requiring high sensitivity

• Limitations: Batch-to-batch variability, potential for higher background

Monoclonal Antibodies (e.g., rabbit monoclonal [EP1934Y]):

• Advantages: Consistent batch-to-batch performance, higher specificity for a single epitope, lower background

• Recommended for: Quantitative studies, experiments requiring high reproducibility, differential detection of protein isoforms

• Limitations: May be less robust to fixation-induced epitope changes

For critical experiments, validation with both antibody types may provide complementary information and strengthen research findings.

What controls should I include when using ALDH7A1 antibodies for immunohistochemistry?

Rigorous controls are essential for immunohistochemistry experiments with ALDH7A1 antibodies:

• Positive Tissue Controls: Liver and kidney tissues consistently show strong ALDH7A1 expression and serve as reliable positive controls

• Negative Controls:

  • Primary antibody omission: Replace primary antibody with antibody diluent

  • Isotype controls: Use same species IgG at the same concentration

  • Secondary antibody only: As demonstrated in ab68192 validation

• Antigen Retrieval Optimization:

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes has been validated

  • Pressure cooker methods may improve antigen accessibility in formalin-fixed tissues

• Dilution Series: Perform titration experiments to determine optimal antibody concentration (typically 1:50 - 1:400 for IHC-P)

• Known Expression Pattern Verification: ALDH7A1 shows cytoplasmic staining with potential mitochondrial localization that should be consistent with published patterns

How can I troubleshoot weak or absent signal in Western blots using ALDH7A1 antibodies?

When encountering signal issues in Western blots for ALDH7A1 detection, consider the following methodological adjustments:

• Sample Preparation Optimization:

  • ALDH7A1 is expressed at moderate levels in most tissues; ensure adequate total protein loading (20-50 μg)

  • Include protease inhibitors during lysis to prevent degradation

  • Validated lysates from liver, kidney, or neuroblastoma cell lines (SH-SY5Y) can serve as positive controls

• Antibody Concentration Adjustment:

  • Increase primary antibody concentration (recommended range: 1:1000 - 1:4000)

  • Extend primary antibody incubation to overnight at 4°C

  • Optimize secondary antibody dilution (typical range: 1:2000 - 1:50000)

• Detection System Enhancement:

  • Switch to more sensitive detection methods (ECL Plus vs. standard ECL)

  • Adjust exposure time (validated examples show exposure times of 3-10 seconds)

  • Consider using HRP-conjugated detection systems for signal amplification

• Protein Transfer Verification:

  • Use reversible staining methods to confirm protein transfer to membrane

  • The predicted molecular weight for ALDH7A1 is 58 kDa, with additional bands at 51-55 kDa representing potential isoforms

• Buffer Optimization:

  • Increase blocking time/concentration to reduce background

  • Add 0.1% Tween-20 to wash buffers to reduce non-specific binding

What approaches are recommended for detecting subcellular localization of ALDH7A1?

ALDH7A1 exhibits complex subcellular distribution patterns that require careful methodological consideration:

• Immunofluorescence Protocols:

  • Fixation: 4% formaldehyde fixation (10-15 minutes) preserves ALDH7A1 epitopes

  • Permeabilization: 0.2% Triton X-100 allows antibody access to intracellular compartments

  • Blocking: 10% normal goat serum reduces background signal

  • Antibody incubation: Overnight at 4°C with dilutions of 1:50 - 1:200

  • Counterstaining: Use established markers for mitochondria (MitoTracker), cytosol, or other organelles for co-localization studies

• Co-localization Analysis:

  • ALDH7A1 has been reported in both cytosolic and mitochondrial compartments

  • Alternative translation initiation sites may lead to different subcellular targeting

  • Advanced confocal microscopy with Z-stack acquisition is recommended for precise localization

• Fractionation Approaches:

  • Complement immunofluorescence with subcellular fractionation

  • Western blot analysis of fractionated cell components can confirm distribution patterns

  • Include compartment-specific markers (e.g., GAPDH for cytosol, COX IV for mitochondria)

How can I optimize ALDH7A1 antibodies for multiplex immunoassays?

For researchers developing multiplex assays incorporating ALDH7A1 detection:

• Antibody Pair Selection:

  • Use validated matched antibody pairs such as 84871-2-PBS (capture) and 84871-3-PBS (detection) for cytometric bead arrays

  • Ensure antibodies recognize non-overlapping epitopes to prevent steric hindrance

• Conjugation Considerations:

  • Select conjugation-ready formats (PBS only, BSA and azide-free) for optimal labeling

  • Validate each conjugated antibody individually before multiplexing

  • Determine optimal antibody concentration post-conjugation

• Cross-Reactivity Testing:

  • Test for cross-reactivity with other targets in your multiplex panel

  • Include appropriate isotype controls for each antibody species/class

  • Validate signal specificity using ALDH7A1 knockdown/knockout samples

• Signal Optimization:

  • Titrate antibody concentrations to achieve comparable signal intensity across targets

  • Adjust incubation times and temperatures to enhance specific binding

  • Consider sequential rather than simultaneous incubation if cross-reactivity is observed

What approaches are recommended for studying ALDH7A1 in the context of oxidative stress research?

Given ALDH7A1's role in metabolizing lipid peroxidation-derived aldehydes and protecting against oxidative stress , researchers should consider:

• Experimental Models:

  • Cell Culture: Induce oxidative stress with H₂O₂, paraquat, or other agents

  • Animal Models: Ischemia-reperfusion, aging, or toxicant exposure models

  • Patient Samples: Tissues from conditions associated with oxidative stress

• Functional Assessment Approaches:

  • Activity Assays: Measure enzymatic activity using betaine aldehyde or lipid peroxidation-derived aldehyde substrates

  • Knockdown/Overexpression: Use siRNA or overexpression constructs to modulate ALDH7A1 levels

  • Inhibition Studies: Apply specific ALDH inhibitors to assess functional contributions

• Downstream Analysis:

  • Combine antibody-based detection with measurement of oxidative stress markers

  • Assess cell viability, ROS levels, and lipid peroxidation in ALDH7A1-modulated systems

  • Investigate protein-protein interactions under stress conditions using co-immunoprecipitation

• Translational Applications:

  • Correlate ALDH7A1 expression/activity with clinical parameters in disease states

  • Examine potential biomarker applications in conditions with underlying oxidative stress

How should I address epitope masking issues in fixed tissue samples?

When working with fixed tissues that may present epitope accessibility challenges:

• Antigen Retrieval Optimization:

  • High-pressure antigen retrieval in citrate buffer (pH 6.0) is recommended for ALDH7A1 detection

  • Standardize the timing (20 minutes) and temperature for consistent results

  • Alternative buffers (EDTA, pH 9.0) may be tested if standard protocols yield weak signals

• Fixation Considerations:

  • Overfixation can permanently mask epitopes; limit fixation time for prospective samples

  • For archival FFPE tissues, extend antigen retrieval time

  • Consider testing alternate antibodies targeting different epitopes if particular samples consistently fail

• Signal Amplification Strategies:

  • Employ biotinylated secondary antibodies with HRP-conjugated streptavidin systems

  • Tyramide signal amplification may enhance detection of low-abundance targets

  • Increase primary antibody concentration (1:50 - 1:200) for challenging samples

What strategies are recommended for quantitative analysis of ALDH7A1 expression?

For researchers requiring precise quantification of ALDH7A1:

• Western Blot Quantification:

  • Include loading controls appropriate for your experimental context (β-actin, GAPDH, total protein staining)

  • Establish a standard curve using recombinant ALDH7A1 protein

  • Use digital image analysis software with appropriate background correction

  • Report relative expression normalized to controls

• Immunohistochemistry Quantification:

  • Develop consistent scoring methods (H-score, percentage positive cells, intensity scales)

  • Use digital pathology platforms for automated quantification

  • Include calibration standards on each slide for inter-slide normalization

  • Report data as continuous variables where possible

• Flow Cytometry Approaches:

  • Ensure appropriate permeabilization for intracellular ALDH7A1 detection

  • Use calibration beads to standardize fluorescence intensity

  • Report data as median fluorescence intensity or percentage positive cells

  • Include appropriate isotype and negative controls

• qPCR Correlation:

  • Complement protein-level data with mRNA quantification

  • Design primers spanning exon-exon junctions for specificity

  • Validate correlations between protein and mRNA levels in your specific system

How do I select the appropriate ALDH7A1 antibody for studies of protein-protein interactions?

When investigating ALDH7A1 interactions with other proteins:

• Epitope Considerations:

  • Select antibodies targeting epitopes unlikely to be involved in protein-protein interactions

  • C-terminal antibodies (e.g., those targeting AA 430-539) may be suitable for many interaction studies

  • Review structural data if available to identify potentially masked regions

• Immunoprecipitation Optimization:

  • Validated antibodies for IP applications include ab236663

  • Use mild lysis conditions to preserve protein complexes (avoid strong detergents)

  • Include appropriate negative controls (IgG from the same species)

  • Confirm specificity of pulled-down complexes with multiple detection methods

• Proximity Ligation Assays:

  • Consider this technique for in situ detection of protein-protein interactions

  • Requires antibodies from different species or directly conjugated antibodies

  • Validate specificity with known interaction partners or negative controls

• Co-localization Studies:

  • Combine with biochemical approaches for stronger evidence

  • Use super-resolution microscopy for precise spatial relationship analysis

  • Quantify co-localization using appropriate statistical methods

What roles does ALDH7A1 play in disease contexts, and how can antibodies help elucidate these mechanisms?

ALDH7A1 has been implicated in several disease processes where antibody-based detection provides valuable insights:

• Pyridoxine-Dependent Epilepsy:

  • Mutations in ALDH7A1 are associated with this disorder

  • Antibodies can help assess expression levels and patterns in patient samples

  • Immunohistochemistry of brain tissue can reveal altered distribution patterns

  • Western blotting can detect truncated or unstable protein variants

• Cancer Research Applications:

  • ALDH7A1 may serve as a cancer stem cell marker in certain malignancies

  • Multiplex immunofluorescence can characterize co-expression with other stem cell markers

  • Tissue microarray analysis can assess expression across tumor subtypes and stages

  • Correlation with clinical outcomes can identify potential prognostic value

• Neurodegenerative Disorders:

  • Given its role in protecting against oxidative stress, ALDH7A1 may be relevant in conditions like Alzheimer's and Parkinson's diseases

  • Antibodies can track expression changes in disease progression

  • Co-staining with markers of neurodegeneration can reveal spatial relationships

• Methodological Approaches:

  • Combine genetic models (knockouts, disease-specific mutations) with antibody detection

  • Correlate protein expression with functional assays and clinical parameters

  • Consider longitudinal studies to track expression changes over disease course

How can I approach ALDH7A1 detection in non-traditional sample types?

As research extends to diverse biological contexts, specialized approaches for ALDH7A1 detection may be required:

• Biofluid Analysis:

  • For cerebrospinal fluid, concentrate samples before Western blotting

  • In serum/plasma, immunoprecipitation may be required before detection

  • Consider developing ELISA approaches with validated antibody pairs

  • Account for potential interference from abundant proteins

• Primary Cell Cultures:

  • Optimize fixation protocols to preserve native epitopes

  • Use lower antibody concentrations (start at 1:200) to minimize background

  • Include tissue-specific markers to confirm cell identity

  • Compare expression patterns with corresponding tissue sections

• 3D Culture Systems:

  • Extend permeabilization times for adequate antibody penetration

  • Use confocal microscopy with Z-stack acquisition for complete visualization

  • Consider cleared tissue approaches for whole-mount immunostaining

  • Validate antibody penetration using known markers with defined distributions

• Tissue-Specific Considerations:

  • High lipid content tissues: Modify extraction buffers to improve protein recovery

  • Connective tissues: Extend protease digestion steps during sample preparation

  • Highly vascularized tissues: Implement perfusion steps to reduce blood contamination