AADAT Antibody

What is AADAT and what role does it play in human metabolism?

AADAT (Aminoadipate aminotransferase) is a metabolic enzyme involved in several critical pathways including lysine biosynthesis, lysine degradation, tryptophan metabolism, and thyroid hormone regulation . Also known by synonyms KAT2, KATII, and KYAT2, AADAT functions as a transaminase that catalyzes the conversion of specific substrates .

Recent research has established AADAT as an enzyme that effectively catalyzes the transamination of thyroid hormones T4 and particularly T3 to their respective pyruvic acid metabolites TK4 and TK3 . This represents an important metabolic pathway for thyroid hormone clearance, complementing the well-established deiodination pathway. AADAT is highly expressed in the liver, gastrointestinal tract, and kidney in humans, suggesting its importance in systemic metabolism .

How does AADAT contribute to thyroid hormone regulation?

AADAT plays a significant role in thyroid hormone metabolism through its enzymatic activity. Functional analyses have demonstrated that:

• AADAT effectively catalyzes the transamination of both T4 and T3 to TK4 and TK3, respectively, with particularly high efficiency for T3 conversion

• These pyruvic acid metabolites (TK3 and TK4) have been detected in urine and bile of experimental models given radio-labeled thyroid hormones

• The enzyme's high expression in liver, kidney, and gastrointestinal tissues positions it as a key contributor to thyroid hormone clearance pathways

Genome-wide association studies have identified that genetic variants affecting AADAT expression impact circulating thyroid hormone levels. Specifically, variants that decrease AADAT transcript levels in thyroid tissue lead to increased circulating FT4 levels, confirming its importance in thyroid hormone homeostasis .

What are the optimal conditions for using AADAT antibodies in Western blot applications?

When using AADAT antibodies for Western blotting, researchers should follow these guidelines for optimal results:

• Dilution Range: Commercial AADAT antibodies typically work best at dilutions of 1:500~1:1000 for Western blotting applications

• Sample Preparation: Use protein extracts from tissues with known high AADAT expression (liver, kidney, intestine) as positive controls

• Expected Molecular Weight: Human AADAT appears at approximately 48 kDa on Western blots

• Buffer System: Standard PBS with pH 7.4 is recommended as the primary buffer system

• Storage Conditions: Store antibodies at -20°C to maintain stability; most AADAT antibodies remain stable for 12 months from receipt date

• Species Reactivity: Verify the specific species reactivity of your AADAT antibody; many are reactive with human AADAT but may have variable cross-reactivity with mouse or rat orthologs

For reproducible results, always include appropriate positive and negative controls and verify consistent protein loading across samples.

In which tissues and cell types is AADAT predominantly expressed?

AADAT shows a tissue-specific expression pattern that researchers should consider when designing experiments:

This expression profile is consistent across studies and provides valuable guidance for selecting appropriate experimental models . The high expression in metabolically active tissues correlates with AADAT's role in amino acid and thyroid hormone metabolism. When conducting immunohistochemistry or tissue-specific studies, these high-expression tissues serve as excellent positive controls for antibody validation.

What experimental models are most appropriate for studying AADAT function?

When selecting experimental models to study AADAT function, researchers should consider:

• Cell Lines:

  • Hepatic cell lines (HepG2, Huh7) for liver-specific functions

  • Renal cell lines (HEK293, RPTEC) for kidney-related studies

  • Intestinal epithelial cells (Caco-2, HT-29) for gastrointestinal research

• Animal Models:

  • Rodent models with tissue expression patterns similar to humans

  • Genetic knockout or knockdown models to study loss-of-function effects

  • Transgenic models with altered AADAT expression to study gain-of-function effects

• Primary Cells:

  • Primary hepatocytes for metabolic studies

  • Primary renal epithelial cells for kidney-specific functions

  • Primary intestinal organoids for digestive tract research

For thyroid hormone metabolism studies specifically, experimental systems should allow for measurement of thyroid hormone transamination activity, using either radiolabeled hormones or sensitive mass spectrometry techniques.

How do genetic variants of AADAT affect thyroid function and disease risk?

Genetic studies have revealed significant associations between AADAT variants and thyroid function:

• Impact on Thyroid Hormone Levels: Genome-wide association studies identified AADAT as one of 109 independent genetic variants associated with thyroid function and dysfunction . Specifically, certain AADAT variants lead to decreased AADAT transcript levels in thyroid tissue, which correlates with increased circulating FT4 levels .

• Disease Risk Association: A genetic risk score incorporating AADAT variants shows significant associations with both overt thyroid disease (including Graves' disease) and subclinical thyroid dysfunction . This suggests AADAT's involvement in thyroid pathophysiology extends beyond normal variation to disease susceptibility.

• Mechanism: The underlying mechanism appears to involve altered metabolism of thyroid hormones through the transamination pathway. Decreased AADAT activity likely leads to reduced clearance of thyroid hormones, particularly T3, affecting systemic thyroid hormone levels .

• Co-localization Evidence: eQTL co-localization studies indicate that the index SNP decreases AADAT transcript levels in the thyroid, mechanistically explaining the association with increased circulating FT4 levels .

These findings position AADAT as both a biomarker and potential therapeutic target in thyroid disease management.

What are the critical steps in validating AADAT antibody specificity?

Proper validation of AADAT antibody specificity requires a multi-faceted approach:

• Positive and Negative Controls:

  • Use tissues with known high AADAT expression (liver, kidney) as positive controls

  • Include AADAT-knockout or knockdown samples as negative controls

  • Test non-expressing tissues to confirm absence of non-specific binding

• Peptide Competition Assays:

  • Pre-incubate antibody with the immunizing peptide (typically a synthetic peptide derived from human AADAT)

  • Observe elimination or significant reduction of signal to confirm specificity

• Multiple Detection Methods:

  • Cross-validate results using different techniques (Western blot, immunohistochemistry, ELISA)

  • Confirm subcellular localization patterns match expected AADAT distribution

• Orthogonal Validation:

  • Compare antibody detection with mRNA expression data

  • Use multiple antibodies targeting different AADAT epitopes

  • Correlate with functional enzyme activity when possible

• Specificity Verification:

  • Test for cross-reactivity with related aminotransferase enzymes

  • Evaluate species cross-reactivity if using the antibody across multiple organisms

Thorough validation ensures experimental results truly reflect AADAT biology rather than artifacts or non-specific interactions.

How can AADAT antibodies be used to investigate the role of AADAT in autoimmune conditions?

Recent research suggests connections between AADAT and autoimmune conditions that can be explored using AADAT antibodies:

• Thyroid Autoimmunity Studies:

  • AADAT variants have been associated with thyroid peroxidase antibody (TPOAb) positivity and Graves' disease

  • Researchers can use AADAT antibodies to examine protein expression in thyroid tissue from autoimmune thyroid disease patients

  • Immunohistochemistry can reveal altered expression patterns in diseased versus healthy thyroid tissue

• Anti-AADAT Autoantibodies:

  • Some studies suggest anti-AADAT autoantibodies may serve as biomarkers in certain autoimmune conditions

  • Researchers can develop assays to detect these autoantibodies in patient sera using purified AADAT as the capture antigen

• Mechanistic Investigations:

  • AADAT antibodies can help examine enzyme localization and expression levels in various immune cells

  • Co-immunoprecipitation using AADAT antibodies may identify novel interaction partners in immune contexts

  • Tissue-specific changes in AADAT expression during autoimmune disease progression can be monitored

• Therapeutic Target Exploration:

  • Neutralizing antibodies against AADAT could be developed to test the effect of enzyme inhibition

  • Expression studies using AADAT antibodies can identify patient subgroups that might benefit from AADAT-targeting therapies

The growing evidence linking AADAT to autoimmune conditions, particularly thyroid autoimmunity, makes this an important area for further investigation using well-validated antibody tools .

What methodological approaches are recommended for studying AADAT's role in thyroid hormone metabolism?

To effectively investigate AADAT's role in thyroid hormone metabolism, researchers should consider these methodological approaches:

• Enzyme Activity Assays:

  • Develop assays to measure the conversion of T3/T4 to TK3/TK4 in various biological samples

  • Use mass spectrometry to detect and quantify thyroid hormone metabolites

  • Compare activity in different tissues and under various physiological conditions

• Protein-Level Analyses:

  • Use validated AADAT antibodies for Western blotting to quantify expression levels

  • Perform immunohistochemistry to localize AADAT in thyroid and peripheral tissues

  • Employ immunoprecipitation to isolate AADAT complexes that may regulate its function

• Genetic Approaches:

  • Study the effects of AADAT variants identified in GWAS on protein expression and activity

  • Develop knockout or knockdown models to assess the impact on thyroid hormone levels

  • Create cell lines expressing AADAT variants to test functional consequences

• Metabolic Profiling:

  • Perform comprehensive metabolic profiling in models with altered AADAT expression

  • Track thyroid hormone metabolites in circulation and tissues

  • Correlate metabolite levels with AADAT expression as detected by antibodies

• Clinical Correlations:

  • Compare AADAT expression in thyroid disease patients versus controls

  • Correlate genetic variants with protein expression and clinical parameters

  • Investigate relationships between AADAT activity and response to thyroid hormone replacement therapy

These approaches leverage the specificity of AADAT antibodies while providing complementary data on enzyme function and physiological significance.

How do protein interactions and post-translational modifications affect AADAT antibody binding?

Protein interactions and post-translational modifications can significantly impact AADAT antibody recognition:

• Protein-Protein Interactions:

  • Binding partners may mask antibody epitopes, reducing detection efficiency

  • Conformational changes induced by protein interactions can expose or conceal epitopes

  • Consider using different lysis conditions to disrupt protein complexes when necessary

• Post-Translational Modifications (PTMs):

  • Phosphorylation, acetylation, or other PTMs may alter epitope recognition

  • Some PTMs are tissue-specific or condition-dependent, leading to variable antibody binding

  • Consider using phosphatase treatment or other enzymatic approaches to remove PTMs when evaluating their impact

• Conformation-Dependent Recognition:

  • Native versus denatured AADAT may be recognized differently by antibodies

  • Some antibodies work better in Western blot (denatured protein) than immunoprecipitation (native protein)

  • Test antibodies under both native and denaturing conditions when possible

• Experimental Considerations:

  • Use multiple antibodies targeting different epitopes to obtain comprehensive detection

  • Consider how sample preparation methods may affect protein modifications

  • Include appropriate controls when studying conditions that might alter PTM status

Understanding these factors is crucial for accurate interpretation of experimental results, particularly when comparing AADAT detection across different physiological or pathological states.

What are the common sources of inconsistent results when using AADAT antibodies?

When faced with inconsistent results using AADAT antibodies, consider these common issues:

• Sample Preparation Problems:

  • Protein degradation during extraction (add protease inhibitors)

  • Insufficient protein denaturation for Western blotting

  • Overfixation masking epitopes in immunohistochemistry

  • Variable expression levels in different tissue regions

• Antibody-Related Factors:

  • Lot-to-lot variability in polyclonal antibodies

  • Antibody degradation due to improper storage

  • Insufficient antibody concentration

  • Epitope specificity issues affecting detection of splice variants

• Technical Variables:

  • Inconsistent transfer efficiency in Western blotting

  • Variable blocking efficiency causing background differences

  • Incubation temperature fluctuations affecting binding kinetics

  • Detection system sensitivity variations

• Biological Considerations:

  • AADAT expression varies with metabolic state

  • Disease conditions may alter post-translational modifications

  • Age, sex, or treatment status may affect expression patterns

  • Species differences when using antibodies across different models

For each potential issue, implement targeted troubleshooting steps and maintain detailed records of experimental conditions to identify patterns in variability.

How can researchers optimize immunoprecipitation protocols for AADAT studies?

Optimizing immunoprecipitation (IP) of AADAT requires attention to several critical parameters:

• Buffer Optimization:

  • Use PBS (pH 7.4) as a base buffer, which matches the storage conditions of many AADAT antibodies

  • Add mild detergents (0.1-0.5% NP-40 or Triton X-100) to maintain protein solubility

  • Include protease inhibitors to prevent degradation during extraction and IP

  • Consider phosphatase inhibitors if studying phosphorylation states

• Antibody Selection and Application:

  • Use antibodies specifically validated for immunoprecipitation

  • Determine optimal antibody amount through titration (typically 2-5 μg per reaction)

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Consider direct antibody conjugation to beads to avoid heavy chain interference in subsequent Western blots

• Incubation Parameters:

  • Compare short (2-4 hours) versus overnight incubations at 4°C

  • Maintain gentle agitation to promote antibody-antigen binding

  • Optimize protein concentration in lysates (typically 0.5-2 mg/ml total protein)

• Washing and Elution:

  • Develop a washing strategy that balances specificity with yield

  • Consider sequential washes with decreasing stringency

  • Compare gentle elution (peptide competition) versus denaturing elution (SDS buffer)

  • Validate eluates by Western blotting with a different AADAT antibody

This optimized protocol can be used to study AADAT interactions with other proteins or to isolate AADAT for activity assays or further characterization.

What controls are essential for immunohistochemistry experiments targeting AADAT?

When performing immunohistochemistry with AADAT antibodies, include these essential controls:

• Positive Tissue Controls:

  • Liver sections (high AADAT expression)

  • Kidney cortex sections (high AADAT expression)

  • Intestinal epithelium sections (high AADAT expression)

• Negative Controls:

  • Primary antibody omission (tests secondary antibody specificity)

  • Isotype control (irrelevant primary antibody of same isotype)

  • Peptide competition (pre-incubation with immunizing peptide)

  • AADAT-knockdown or knockout tissue (if available)

• Procedural Controls:

  • Antigen retrieval optimization series

  • Antibody dilution series to determine optimal concentration

  • Varying incubation times to optimize signal-to-noise ratio

• Validation Controls:

  • Parallel Western blotting of the same tissues

  • Correlation with known mRNA expression patterns

  • Comparison with alternative AADAT antibodies

A comprehensive control strategy ensures that staining patterns accurately represent AADAT distribution rather than technical artifacts or non-specific binding.

How can researchers correlate AADAT enzyme activity with antibody-based detection methods?

Correlating AADAT enzyme activity with protein detection provides valuable insights into functional relationships:

• Sequential Analysis Approach:

  • Split samples for parallel protein detection and activity measurement

  • Use Western blotting with AADAT antibodies to quantify protein levels

  • Measure enzyme activity through:

    • Transamination of T3/T4 to TK3/TK4 using chromatography/mass spectrometry

    • Conversion of kynurenine to kynurenic acid (KAT activity)

    • Alpha-aminoadipate transamination assays

  • Correlate activity levels with protein expression across samples

• Combined Activity-Detection Methods:

  • Immunoprecipitate AADAT using validated antibodies

  • Measure enzyme activity in the immunoprecipitate

  • Verify pulled-down protein by Western blotting

  • Compare activity per unit of immunoprecipitated protein across conditions

• Tissue-Specific Correlations:

  • Perform immunohistochemistry to localize AADAT in tissue sections

  • Prepare homogenates from adjacent tissue sections for activity assays

  • Create activity maps that can be compared with expression patterns

• Genetic Manipulation Studies:

  • Create AADAT overexpression or knockdown models

  • Verify protein level changes using antibody-based methods

  • Measure corresponding changes in enzymatic activity

  • Establish dose-response relationships between expression and function

This multi-faceted approach helps distinguish between inactive and active AADAT pools and provides insight into post-translational regulation that may not be evident from protein measurements alone.

What are the recommended storage and handling conditions for maintaining AADAT antibody stability?

To maintain AADAT antibody stability and performance, follow these storage and handling recommendations:

• Storage Temperature:

  • Store antibodies at -20°C as recommended by manufacturers

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

  • Working stocks can be kept at 4°C for up to two weeks

• Buffer Conditions:

  • Most AADAT antibodies are stable in PBS, pH 7.4 with 0.02% sodium azide

  • Avoid introducing contaminants into stock solutions

  • Do not add detergents or other additives unless specified by the manufacturer

• Physical Handling:

  • Minimize exposure to light, particularly for fluorophore-conjugated antibodies

  • Avoid vigorous shaking or vortexing that may denature antibody proteins

  • Use low-protein binding tubes for dilute antibody solutions

• Stability Considerations:

  • Most AADAT antibodies remain stable for 12 months from receipt date when properly stored

  • Monitor performance over time with consistent positive controls

  • Consider refreshing antibody stocks for critical experiments if stored for extended periods

• Working Solution Preparation:

  • Prepare fresh dilutions for each experiment

  • Use high-quality, filtered buffers for dilutions

  • Allow refrigerated antibodies to equilibrate to room temperature before opening to prevent condensation

Proper storage and handling significantly impact antibody performance and experimental reproducibility, making these considerations essential for reliable AADAT research.