ALDH12A1 Antibody

What is ALDH12A1 and what cellular functions does it regulate?

ALDH12A1 (Aldehyde dehydrogenase family 12 member A1), also known as delta-1-pyrroline-5-carboxylate dehydrogenase 12A1 or P5CDH, is a mitochondrial enzyme involved in proline metabolism . It catalyzes the conversion of P5C to glutamate, which is an essential step in proline catabolism. By preventing P5C accumulation, ALDH12A1 protects cells from oxidative stress and programmed cell death. This protein is structurally related to other aldehyde dehydrogenase family members, which are involved in numerous metabolic pathways including alcohol metabolism, retinoic acid synthesis, and detoxification processes .

The ALDH family, to which ALDH12A1 belongs, includes important enzymes like ALDH1A1 and ALDH1A2 that serve critical functions in oxidative pathways and retinoid metabolism . While ALDH1A1 is primarily cytosolic and involved in alcohol metabolism , and ALDH1A2 catalyzes the synthesis of retinoic acid from retinaldehyde , ALDH12A1 specifically functions in the proline metabolic pathway.

What are the recommended applications for ALDH12A1 antibody?

The ALDH12A1 antibody is validated for multiple experimental applications including:

These applications enable researchers to detect and quantify ALDH12A1 protein expression, localize it within cellular compartments, and study its interactions with other proteins . When designing experiments, researchers should consider the monoclonal nature of the antibody and its specific reactivity pattern.

What is the proper storage and handling protocol for ALDH12A1 antibody?

ALDH12A1 antibody should be stored at -20°C, where it remains stable for approximately 12 months from the date of receipt . To maintain antibody integrity:

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• Thaw aliquots at room temperature and briefly centrifuge before use

• Keep the antibody on ice during experimental procedures

• Return to -20°C promptly after use

• Monitor for signs of degradation such as precipitation or loss of activity

These storage conditions are similar to those for other antibodies in the ALDH family, which also require careful handling to maintain their specificity and reactivity in experimental applications .

How should ALDH12A1 antibody be validated for specificity in my experimental system?

Validating antibody specificity is crucial for accurate results. For ALDH12A1 antibody, implement these validation approaches:

• Positive and negative controls: Include known ALDH12A1-expressing samples (positive control) and samples with low or no expression (negative control)

• Knock-down/knock-out verification: If possible, use ALDH12A1 knock-down or knock-out samples to confirm specificity

• Blocking peptide assay: Pre-incubate the antibody with the immunizing peptide to demonstrate signal reduction

• Cross-reactivity assessment: Test the antibody against recombinant ALDH12A1 and related family members to confirm specificity

• Molecular weight verification: Confirm detection at the expected molecular weight (~55-60 kDa) on Western blots

Similar validation approaches are used for other ALDH family antibodies , but must be specifically adapted for ALDH12A1 given its unique expression patterns and cellular localization.

What are the optimal conditions for Western blot detection of ALDH12A1?

For optimal Western blot detection of ALDH12A1:

• Sample preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Include reducing agents (β-mercaptoethanol or DTT) in sample buffer

  • Heat samples at 95°C for 5 minutes to denature proteins

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels

  • Transfer to PVDF membranes (rather than nitrocellulose) for better protein retention

  • Transfer at low voltage (30V) overnight at 4°C for complete transfer of mitochondrial proteins

• Blocking and antibody incubation:

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

  • Incubate with primary ALDH12A1 antibody (1:1000 dilution) overnight at 4°C

  • Use secondary antibody at 1:5000-1:10000 dilution for 1 hour at room temperature

• Detection optimization:

  • Use enhanced chemiluminescence detection systems

  • Optimize exposure times to prevent signal saturation

This methodology draws on established protocols for other aldehyde dehydrogenase family members , adapted specifically for ALDH12A1 detection.

How can ALDH12A1 antibody be used in multimodal single-cell analysis?

Incorporating ALDH12A1 antibody into multimodal single-cell analysis requires careful optimization to balance signal-to-background ratio while maintaining cell viability:

• Antibody titration: Perform systematic titration experiments (testing concentrations from 0.1-10 μg/mL) to determine optimal concentration that maximizes specific signal while minimizing background

• Background assessment: Quantify background signal in empty droplets versus cell-containing droplets when using ALDH12A1 antibody in single-cell platforms

• Panel design considerations:

  • Combine with complementary markers based on expected co-expression patterns

  • Account for epitope abundance when optimizing antibody concentrations

  • Validate specificity in the context of multiparameter analysis

• Data analysis approaches:

  • Apply background correction algorithms specific to oligo-conjugated antibodies

  • Use appropriate normalization strategies for integrated analysis with transcriptomic data

This approach builds on recent advances in multimodal single-cell technologies that have revealed the importance of antibody optimization for accurate protein detection alongside RNA measurements .

What are the key considerations when using ALDH12A1 antibody for studying proline metabolism in disease models?

When investigating proline metabolism using ALDH12A1 antibody in disease models:

• Context-dependent expression: Evaluate ALDH12A1 expression across different tissues and disease states, as expression patterns may vary significantly

• Functional readouts: Pair antibody-based detection with functional assays that measure:

  • P5C accumulation

  • Reactive oxygen species (ROS) levels

  • Cell death markers

  • Glutamate production

• Experimental design for metabolic studies:

  • Include metabolic stress conditions (nutrient deprivation, hypoxia)

  • Consider time-course analyses to capture dynamic regulation

  • Compare ALDH12A1 expression with related enzymes in proline metabolism

• Tissue-specific considerations:

  • Optimize fixation protocols for different tissue types

  • Adjust antibody concentrations based on expected expression levels

  • Account for potential cross-reactivity with other ALDH family members

These approaches are adapted from methodologies used to study related ALDH family proteins in disease contexts , with specific modifications for ALDH12A1's role in proline metabolism.

What are common issues with ALDH12A1 antibody signal detection and how can they be resolved?

These troubleshooting approaches draw on general antibody optimization principles and have been adapted to address specific challenges with ALDH family proteins .

How can I differentiate between ALDH12A1 and other ALDH family members in my experiments?

Distinguishing ALDH12A1 from other ALDH family members requires careful experimental design:

• Antibody selection strategy:

  • Verify the epitope region targeted by the antibody

  • Choose antibodies raised against unique regions with minimal sequence homology to other ALDH proteins

  • Consider using antibodies from different host species for co-localization studies

• Comparative analysis approach:

  • Include parallel experiments with antibodies against related family members (ALDH1A1, ALDH1A2)

  • Compare molecular weights: ALDH12A1 versus ALDH1A1 (~55kDa) and ALDH1A2

  • Assess subcellular localization (ALDH12A1 is primarily mitochondrial, while ALDH1A1 is cytosolic)

• Validation techniques:

  • Use RNA interference targeting specific ALDH family members

  • Employ immunoprecipitation followed by mass spectrometry for definitive identification

  • Consider recombinant protein controls for each ALDH family member

• Expression pattern analysis:

  • Compare tissue-specific expression patterns of different ALDH family members

  • Utilize differential expression in response to specific stimuli

This approach leverages information about the distinct properties of ALDH family members to ensure accurate identification of ALDH12A1 in experimental systems .

How can ALDH12A1 antibody be integrated into high-throughput screening platforms?

Integrating ALDH12A1 antibody into high-throughput screening (HTS) platforms represents an emerging research direction with several technical considerations:

• Automation-compatible protocols:

  • Develop fixed-plate immunofluorescence assays using ALDH12A1 antibody

  • Optimize for 384- or 1536-well plate formats

  • Establish robotics-compatible washing and incubation steps

• Readout optimization:

  • Develop quantitative image analysis algorithms specific to ALDH12A1 subcellular localization

  • Implement machine learning for automated phenotype classification

  • Create reference standards for normalization across screening batches

• Multiplex detection strategies:

  • Combine ALDH12A1 antibody with markers of cellular stress and metabolism

  • Design orthogonal assays to confirm hits (enzymatic activity, metabolite measurements)

  • Develop oligonucleotide-conjugated ALDH12A1 antibodies for sequence-based readouts

• Validation pipeline:

  • Establish confirmation protocols for screening hits

  • Include counter-screens to eliminate false positives

  • Design secondary assays to assess functional impacts on proline metabolism

These approaches build on recent advances in antibody-based high-throughput screening methodologies and single-cell technologies while addressing the specific requirements for accurate ALDH12A1 detection.

What are emerging applications of ALDH12A1 antibody in studying metabolic dysfunction and cell death pathways?

Emerging research applications for ALDH12A1 antibody include:

• Investigation of metabolic stress responses:

  • Monitor ALDH12A1 expression changes during nutrient deprivation

  • Correlate ALDH12A1 levels with mitochondrial function markers

  • Examine regulation of ALDH12A1 in response to oxidative stress

• Cell death pathway analysis:

  • Track ALDH12A1 localization during apoptosis initiation

  • Study protective effects against P5C-induced cytotoxicity

  • Investigate interactions with cell death regulators

• Metabolic disease models:

  • Compare ALDH12A1 expression in normal versus diseased tissues

  • Analyze post-translational modifications in pathological states

  • Explore connections between proline metabolism and energy homeostasis

• Therapeutic target validation:

  • Assess ALDH12A1 modulation in response to candidate compounds

  • Develop proximity-based assays to identify ALDH12A1 interactors

  • Screen for small molecule regulators of ALDH12A1 activity

These emerging applications extend our understanding of ALDH12A1's role beyond basic enzymatic function, connecting it to broader cellular processes and potential therapeutic interventions, similar to how other ALDH family members have been studied in various disease contexts .