ALDH6A1 Antibody

What is ALDH6A1 and what is its biological significance?

ALDH6A1 (Aldehyde Dehydrogenase 6 Family, Member A1) is a member of the aldehyde dehydrogenase family of proteins. This enzyme plays a crucial role in the valine and pyrimidine catabolic pathways . It is also known as Methylmalonate-Semialdehyde Dehydrogenase [acylating], which is localized in the mitochondria. The protein has a calculated molecular weight of approximately 57.8 kDa (535 amino acids) . Understanding ALDH6A1's function is important for research in metabolic disorders and potential connections to disease states where these pathways may be dysregulated.

What species reactivity can I expect from commercially available ALDH6A1 antibodies?

Available ALDH6A1 antibodies demonstrate reactivity across several mammalian species. Most commonly, these antibodies show confirmed reactivity with human samples, while many also cross-react with mouse and rat tissues . For example, Proteintech's 20452-1-AP antibody (rabbit polyclonal) has been tested and confirmed to react with human, mouse, and rat samples in multiple applications including Western blot and immunohistochemistry . When selecting an antibody for your research, verify the validated species reactivity from the manufacturer's data to ensure compatibility with your experimental model.

What are the differences between polyclonal and monoclonal ALDH6A1 antibodies, and when should each be used?

The choice between polyclonal and monoclonal antibodies depends on your specific research requirements:

Polyclonal ALDH6A1 antibodies:

• Recognize multiple epitopes on the ALDH6A1 protein

• Examples include Proteintech's 20452-1-AP (rabbit host) and Abbexa's rabbit polyclonal antibody

• Advantage: Higher sensitivity due to binding multiple epitopes

• Best used for: Initial protein detection, applications requiring strong signal (IHC, WB)

• May have higher background in some applications

Monoclonal ALDH6A1 antibodies:

• Recognize a single epitope on the ALDH6A1 protein

• Examples include antibodies-online's ABIN659006 (mouse host, clone 147CT8-3-4)

• Advantage: Higher specificity and consistency between lots

• Best used for: Precise epitope targeting, quantitative applications

• Typically have lower background in immunofluorescence applications

When designing experiments requiring high reproducibility or where background is a concern, monoclonal antibodies may be preferable. For applications where signal strength is paramount, polyclonal antibodies often provide better results.

What are the validated applications for ALDH6A1 antibodies and their recommended dilutions?

ALDH6A1 antibodies have been validated across multiple experimental applications with specific recommended dilution ranges for optimal results:

For optimal results, it is recommended to titrate the antibody concentration in your specific experimental system as sensitivity may vary depending on sample type, preparation method, and detection system used .

What is the recommended protocol for Western blot detection of ALDH6A1?

A standard protocol for Western blot detection of ALDH6A1 should include the following steps:

• Sample preparation:

  • Prepare protein lysates from tissues or cells of interest (validated sources include liver, kidney, heart tissues, and HEK-293 cells)

  • Include appropriate positive controls (e.g., rat liver tissue, mouse heart tissue)

• Gel electrophoresis and transfer:

  • Separate proteins using SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane

• Antibody incubation:

  • Block membrane with appropriate blocking buffer

  • Incubate with primary ALDH6A1 antibody at recommended dilution (e.g., 1:3000-1:12000 for Proteintech 20452-1-AP)

  • Incubate overnight at 4°C for optimal results

  • Wash thoroughly

  • Incubate with species-appropriate secondary antibody

• Detection:

  • Develop using chemiluminescence or other detection methods

  • Expected band size: approximately 58 kDa

For specific detailed protocols, manufacturers often provide optimized protocols for their antibodies that may improve results .

What are the recommended protocols for immunohistochemistry using ALDH6A1 antibodies?

For optimal immunohistochemistry results with ALDH6A1 antibodies:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded tissue sections

  • Validated tissues include human liver, human kidney, and human hepatocirrhosis tissue

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat-mediated retrieval methods are typically most effective

• Antibody incubation:

  • Dilute primary antibody appropriately (e.g., 1:150-1:600 for Proteintech 20452-1-AP or 1:10-1:50 for Abbexa's antibody )

  • Incubate overnight at 4°C or as recommended by manufacturer

  • Use appropriate detection system (e.g., HRP-conjugated secondary antibody and DAB)

• Counterstaining and mounting:

  • Counterstain with hematoxylin

  • Dehydrate and mount with permanent mounting medium

The protocol should be optimized for each specific tissue type and fixation method. Some tissues may require extended antigen retrieval or different dilutions to achieve optimal staining.

How should researchers approach troubleshooting non-specific binding or high background with ALDH6A1 antibodies?

When encountering non-specific binding or high background issues with ALDH6A1 antibodies, consider these systematic troubleshooting approaches:

• For Western blotting:

  • Increase blocking time and/or concentration of blocking agent

  • Reduce primary antibody concentration (try higher dilutions like 1:10000-1:12000)

  • Increase wash steps duration and number

  • Ensure fresh transfer buffers and blocking solutions

  • Consider switching from milk to BSA-based blocking (or vice versa)

  • Use a monoclonal antibody instead of polyclonal if background persists

• For immunohistochemistry/immunofluorescence:

  • Optimize antigen retrieval conditions (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Dilute antibody further (e.g., 1:600 instead of 1:150)

  • Include an avidin/biotin blocking step if using biotin-based detection

  • Use more stringent washing conditions

  • Consider image acquisition settings (reduce exposure/gain)

• General approaches:

  • Validate antibody specificity using positive and negative controls

  • For difficult tissues, include isotype controls to identify non-specific binding

  • Consider alternative antibody clones if persistent issues occur

What are the critical storage and handling conditions for maintaining ALDH6A1 antibody activity?

Proper storage and handling of ALDH6A1 antibodies is essential for maintaining their activity and ensuring reproducible results:

• Long-term storage:

  • Store at -20°C for maximum stability (up to one year)

  • Most ALDH6A1 antibodies are supplied in a stabilizing buffer containing glycerol (typically 50%) that prevents freezing at -20°C

• Short-term storage:

  • For frequent use, store at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles as they can degrade antibody performance

• Working aliquots:

  • For antibodies without glycerol, prepare small working aliquots to avoid repeated freeze-thaw cycles

  • For glycerol-containing antibodies (like Proteintech's 20452-1-AP), aliquoting is unnecessary for -20°C storage

• Buffer composition:

  • Most ALDH6A1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Some smaller volume preparations (20μl) may contain 0.1% BSA for additional stability

• Handling precautions:

  • Always centrifuge antibody vials briefly before opening to collect all liquid

  • Use sterile techniques when handling to prevent contamination

  • Return to recommended storage conditions promptly after use

Following these guidelines will help maintain antibody performance throughout your research project timeline.

How can researchers validate ALDH6A1 antibody specificity for their experimental systems?

Comprehensive validation of ALDH6A1 antibody specificity is crucial for generating reliable research data:

• Positive control tissues/cells:

  • Use validated positive controls including:

    • Rat and mouse liver tissue

    • Mouse heart tissue

    • Mouse and rat kidney tissue

    • HEK-293 cells

  • Expected molecular weight detection at approximately 58 kDa

• Negative controls:

  • Include isotype controls (same host species, same immunoglobulin class, but non-specific target)

  • Consider using ALDH6A1 knockout or knockdown models if available

  • ALDH6A1-low expressing cell lines as comparative controls

• Cross-validation methods:

  • Compare results across multiple detection methods (e.g., WB, IHC, IF)

  • Use multiple antibodies targeting different epitopes of ALDH6A1

  • Correlate protein detection with mRNA expression data

  • Consider peptide blocking experiments with the immunogen

• Application-specific validations:

  • For WB: Confirm single band at expected molecular weight

  • For IHC/IF: Compare staining pattern with known subcellular localization (mitochondrial)

  • Use appropriate positive and negative tissue controls for each application

Thorough validation not only ensures experimental reliability but can also help troubleshoot unexpected results or identify novel expression patterns in your specific research context.

What considerations should be made when using ALDH6A1 antibodies for co-localization studies?

When designing co-localization experiments with ALDH6A1 antibodies, consider these critical factors:

• Subcellular localization context:

  • ALDH6A1 is primarily localized to mitochondria (as indicated by its full name: Methylmalonate-Semialdehyde Dehydrogenase [acylating], Mitochondrial)

  • Consider co-staining with established mitochondrial markers for validation

• Antibody compatibility:

  • Select ALDH6A1 antibodies from different host species than other target proteins

  • For example, use mouse monoclonal ALDH6A1 antibody (e.g., clone 147CT8-3-4) with rabbit antibodies against other proteins

  • Alternatively, use directly conjugated antibodies if available

• Optimal fixation methods:

  • For mitochondrial proteins, 4% paraformaldehyde fixation is often preferred

  • Consider mild permeabilization methods to preserve mitochondrial structure

• Imaging considerations:

  • Use confocal microscopy for precise co-localization analysis

  • Apply appropriate controls for spectral bleed-through

  • Consider super-resolution techniques for detailed mitochondrial studies

• Quantitative analysis:

  • Employ established co-localization analysis methods (Pearson's correlation, Manders' coefficients)

  • Include appropriate positive and negative co-localization controls

How can researchers optimize ALDH6A1 antibody-based immunoprecipitation protocols?

For successful immunoprecipitation of ALDH6A1 and associated proteins:

• Antibody selection:

  • Choose antibodies validated or recommended for immunoprecipitation

  • Polyclonal antibodies often perform better for IP due to recognition of multiple epitopes

  • Ensure the antibody recognizes the native (non-denatured) form of ALDH6A1

• Sample preparation:

  • Use gentle lysis buffers to preserve protein-protein interactions

  • For mitochondrial proteins like ALDH6A1, consider mitochondrial isolation before lysis

  • Include protease inhibitors and phosphatase inhibitors if studying post-translational modifications

• Pre-clearing step:

  • Implement a pre-clearing step with beads alone to reduce non-specific binding

  • Use the same species normal IgG as a negative control

• Optimization strategies:

  • Adjust antibody amounts (typically 1-5 μg per reaction)

  • Optimize incubation times and temperatures (4°C overnight often yields best results)

  • Consider crosslinking antibody to beads to prevent antibody contamination in eluted samples

• Validation of results:

  • Confirm successful IP by Western blot using a different ALDH6A1 antibody targeting a different epitope

  • Include input, unbound, and IP fractions in analysis

  • Consider mass spectrometry to identify novel interacting partners

What approaches should be considered when using ALDH6A1 antibodies for flow cytometry?

When implementing flow cytometry protocols with ALDH6A1 antibodies:

• Protocol optimization:

  • Begin with recommended dilutions (e.g., 1:10-1:50 for Abbexa's antibody)

  • Optimize fixation and permeabilization methods for intracellular/mitochondrial proteins

  • Consider using specialized permeabilization kits for mitochondrial proteins

• Controls:

  • Include appropriate isotype controls matched to primary antibody

  • Use ALDH6A1-high and ALDH6A1-low expressing cells as biological controls

  • Include single-stained samples for compensation when multiplexing

• Sample preparation considerations:

  • For mitochondrial proteins, mild fixation and permeabilization are critical

  • Consider cell cycle phase when analyzing, as mitochondrial content can vary

• Analysis approaches:

  • Gate appropriately based on forward/side scatter to exclude debris and doublets

  • Consider co-staining with mitochondrial markers to verify specificity

  • For quantitative analysis, use median fluorescence intensity rather than percent positive

• Troubleshooting:

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

  • If high background occurs, increase washing steps or dilute antibody further

  • Consider secondary antibody amplification systems for improved sensitivity

These specialized approaches provide methodological frameworks that can be adapted to specific research questions involving ALDH6A1 in various cellular and physiological contexts.

How should researchers interpret variations in ALDH6A1 expression patterns across different tissues?

When analyzing ALDH6A1 expression across tissues:

• Expected tissue distribution patterns:

  • High expression is typically observed in liver and kidney tissues

  • Moderate expression in heart tissue

  • Expression may vary in pathological tissues (e.g., hepatocirrhosis)

• Data normalization approaches:

  • Normalize to appropriate housekeeping proteins based on tissue type

  • Consider multiple normalization controls for cross-tissue comparisons

  • When comparing pathological vs. normal tissues, validate that housekeeping gene expression is not altered

• Subcellular localization interpretation:

  • ALDH6A1 should predominantly show mitochondrial localization

  • Changes in subcellular distribution may indicate altered protein function or stress responses

  • Consider co-staining with mitochondrial markers to verify localization

• Expression level variations:

  • Correlate protein levels with tissue metabolic activity

  • Consider tissue-specific roles in valine and pyrimidine metabolism

  • Account for potential isoforms or post-translational modifications

• Pathological context:

  • Interpret changes in expression in relation to metabolic alterations in disease states

  • Consider correlation with other metabolic enzymes in the same pathway

What considerations should be made when analyzing ALDH6A1 in disease models or clinical samples?

For research involving disease models or clinical specimens:

• Disease relevance context:

  • ALDH6A1 involvement in metabolic pathways suggests potential roles in:

    • Metabolic disorders

    • Neurodegenerative diseases

    • Cancer metabolism

    • Mitochondrial dysfunction disorders

• Sample handling considerations:

  • For clinical samples, standardize collection and processing methods

  • Consider cold ischemia time effects on mitochondrial proteins

  • Implement appropriate preservation methods for maintaining protein integrity

• Analytical approaches:

  • Compare expression levels with established clinical parameters

  • Consider correlation with metabolic biomarkers

  • Implement multi-parameter analysis to identify disease-specific patterns

• Controls and reference ranges:

  • Establish normal reference ranges from appropriate control samples

  • Consider age, sex, and tissue-specific variations

  • Use appropriate disease controls when available

• Interpretation frameworks:

  • Distinguish between causative changes and compensatory responses

  • Consider pathway analysis rather than focusing solely on ALDH6A1

  • Correlate protein changes with functional metabolic outcomes when possible

How can researchers effectively analyze post-translational modifications of ALDH6A1?

For studying post-translational modifications (PTMs) of ALDH6A1:

• Common PTMs to consider:

  • Phosphorylation sites that may regulate enzymatic activity

  • Acetylation (common in mitochondrial proteins)

  • Ubiquitination (affecting protein turnover)

  • Oxidative modifications (relevant to mitochondrial function)

• Experimental approaches:

  • Use phospho-specific or modification-specific antibodies if available

  • Consider enrichment methods for modified proteins before analysis

  • Implement 2D gel electrophoresis to separate modified forms

  • Use mass spectrometry for comprehensive PTM mapping

• Data analysis strategies:

  • Compare modification patterns across physiological and pathological conditions

  • Correlate modifications with enzymatic activity measurements

  • Use bioinformatic prediction tools to identify potential modification sites

• Validation methods:

  • Mutate predicted modification sites to confirm functional relevance

  • Use site-specific antibodies to verify modification presence

  • Correlate with known regulators of the identified modifications

• Physiological context interpretation:

  • Consider how modifications affect enzyme activity, stability, or localization

  • Relate modifications to metabolic state or stress responses

  • Evaluate conservation of modification sites across species to assess functional importance

By implementing these analytical frameworks, researchers can derive meaningful insights from ALDH6A1 studies and place their findings within broader physiological and pathological contexts.

How can ALDH6A1 antibodies be effectively used in multiplex immunofluorescence studies?

For advanced multiplex immunofluorescence applications:

• Antibody panel design:

  • Select ALDH6A1 antibodies with minimal cross-reactivity to other targets

  • Consider using directly conjugated primary antibodies when available

  • For indirect detection, select primaries from different host species

• Technical optimization:

  • If using tyramide signal amplification (TSA) methods, determine optimal antibody dilution and amplification time

  • Implement appropriate spectral unmixing if using closely spaced fluorophores

  • Consider sequential staining protocols for challenging combinations

• Controls for multiplex analysis:

  • Include single-stain controls for each antibody

  • Use isotype controls for each species

  • Include biological positive and negative controls for each target

• Advanced analysis approaches:

  • Implement supervised machine learning for pattern recognition

  • Consider spatial analysis to identify co-expression patterns

  • Use quantitative image analysis software for objective assessment

• Result interpretation:

  • Focus on co-expression patterns in relation to cellular phenotypes

  • Consider subcellular localization patterns for each marker

  • Relate findings to functional metabolic pathways involving ALDH6A1

What considerations should be made when implementing ALDH6A1 antibodies in high-throughput screening applications?

For high-throughput screening with ALDH6A1 antibodies:

• Assay development:

  • Select antibodies with consistent lot-to-lot performance

  • Consider monoclonal antibodies for higher reproducibility

  • Validate in small-scale experiments before scaling up

• Automation compatibility:

  • Determine antibody stability under automated handling conditions

  • Optimize protocols for microplate-based formats

  • Implement quality control checkpoints throughout the workflow

• Data normalization approaches:

  • Develop robust internal controls for plate-to-plate normalization

  • Consider positional effects in plate-based assays

  • Implement appropriate statistical methods for large dataset analysis

• Screening specific considerations:

  • Balance sensitivity and specificity requirements

  • Determine appropriate positive and negative controls for hit identification

  • Implement secondary validation assays for primary hits

• Data analysis frameworks:

  • Develop clear criteria for hit selection

  • Implement appropriate statistical methods for minimizing false positives/negatives

  • Consider machine learning approaches for complex phenotype identification

How should researchers approach using ALDH6A1 antibodies in tissue microarray applications?

For tissue microarray (TMA) studies with ALDH6A1 antibodies:

• TMA design considerations:

  • Include appropriate positive control tissues (liver, kidney)

  • Incorporate normal and pathological tissues for comparison

  • Consider replicates from different regions of the same sample

• Staining protocol optimization:

  • Validate antibody performance on whole sections before TMA application

  • Optimize antigen retrieval for the specific fixation used in TMA preparation

  • Consider automated staining platforms for consistency

• Analysis approaches:

  • Develop standardized scoring methods (e.g., H-score, Allred score)

  • Consider digital pathology and automated image analysis

  • Implement training sets for consistent scoring

• Quality control measures:

  • Include control tissues in each TMA block

  • Monitor staining consistency across multiple TMA slides

  • Implement inter-observer validation for scoring

• Data interpretation frameworks:

  • Correlate expression patterns with clinicopathological parameters

  • Consider survival analysis when appropriate

  • Implement multivariate analysis to identify independent associations

These emerging applications represent cutting-edge approaches for utilizing ALDH6A1 antibodies in complex experimental designs and high-dimensional data analysis scenarios.

What novel applications are emerging for ALDH6A1 antibodies in metabolic disease research?

Emerging applications in metabolic disease research include:

• Metabolic flux analysis:

  • Using ALDH6A1 antibodies to track enzyme localization during metabolic adaptations

  • Correlating ALDH6A1 expression with metabolomic profiles

  • Investigating regulatory mechanisms in response to nutritional stress

• Disease biomarker development:

  • Evaluating ALDH6A1 as a potential biomarker for mitochondrial dysfunction

  • Investigating expression changes in metabolic syndrome and diabetes

  • Exploring connections to branched-chain amino acid metabolism disorders

• Therapeutic target validation:

  • Using antibodies to validate ALDH6A1 as a potential drug target

  • Screening for compounds that modulate ALDH6A1 expression or activity

  • Developing companion diagnostics for metabolic disease therapeutics

• Single-cell applications:

  • Implementing ALDH6A1 antibodies in single-cell protein analysis

  • Investigating cell-to-cell variability in metabolic enzyme expression

  • Correlating with single-cell transcriptomics data

• Translational research approaches:

  • Bridging preclinical models with clinical specimens

  • Developing standardized assays for clinical research applications

  • Exploring pharmacodynamic biomarker potential

How can researchers integrate ALDH6A1 antibody-based data with other -omics approaches?

For multi-omics integration strategies:

• Proteomics integration:

  • Correlate antibody-based ALDH6A1 quantification with mass spectrometry data

  • Investigate protein-protein interaction networks using IP-MS approaches

  • Explore post-translational modifications through targeted proteomics

• Transcriptomics correlation:

  • Compare protein expression patterns with mRNA expression data

  • Investigate potential post-transcriptional regulation mechanisms

  • Identify splice variants that may affect antibody recognition

• Metabolomics connections:

  • Correlate ALDH6A1 expression with metabolite profiles, particularly those in valine and pyrimidine pathways

  • Investigate metabolic pathway flux in relation to enzyme expression

  • Study the impact of ALDH6A1 modulation on the broader metabolome

• Multi-omics data analysis frameworks:

  • Implement pathway enrichment analysis across multiple data types

  • Develop integrated visualization approaches

  • Consider machine learning for pattern recognition across diverse datasets

• Functional validation approaches:

  • Use antibody data to guide functional studies

  • Connect protein expression to enzymatic activity measurements

  • Validate computational predictions with targeted experiments

By implementing these advanced research approaches, investigators can maximize the value of ALDH6A1 antibodies in complex, multi-dimensional studies that address fundamental biological questions and translational research challenges.