OAT Antibody

What is OAT and why is it important in research?

Ornithine aminotransferase (OAT) is a mitochondrial enzyme that catalyzes the reversible conversion of ornithine to glutamate semialdehyde. This 48 kDa protein plays a crucial role in amino acid metabolism, particularly in the urea cycle and proline biosynthesis. The importance of OAT in research stems from its involvement in several metabolic disorders and its potential role as a biomarker in various pathological conditions. The study of OAT using specific antibodies enables researchers to investigate protein expression levels, cellular localization, and functional alterations in different experimental models and human tissues .

What applications are most suitable for OAT antibodies?

OAT antibodies demonstrate utility across multiple laboratory techniques. Based on validation data, these antibodies perform effectively in:

• Western Blot (WB): Detects OAT protein in cell lysates and tissue homogenates with high specificity. Recommended dilutions typically range from 1:5000 to 1:50000 .

• Immunohistochemistry (IHC): Enables visualization of OAT distribution in tissue sections with optimal dilutions between 1:50 and 1:500 .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Reveals subcellular localization of OAT in cultured cells at dilutions of 1:200 to 1:800 .

• ELISA: Provides quantitative detection of OAT in solution .

• Flow Cytometry: Allows assessment of OAT expression in individual cells .

The choice of application should be guided by the specific research question, with experimental conditions optimized for each system to achieve optimal results .

What species reactivity can be expected with OAT antibodies?

Most commercially available OAT antibodies demonstrate cross-reactivity with human, mouse, and rat samples. This multi-species reactivity has been confirmed through various application tests including Western blot, immunohistochemistry, and immunofluorescence . Experimentally validated reactivity includes:

• Human samples: Confirmed in cell lines such as K-562, HeLa, and human liver cancer tissue .

• Mouse samples: Verified in mouse colon and lung tissues .

• Rat samples: Demonstrated in rat colon and lung tissues .

This cross-species reactivity makes these antibodies versatile tools for comparative studies across different model organisms, although researchers should always perform validation tests in their specific experimental systems .

What are the recommended dilutions and protocols for OAT antibody applications?

Optimal dilutions for OAT antibodies vary by application technique and specific antibody clone. Based on validated protocols:

All applications require sample-dependent optimization, as cellular or tissue expression levels may vary significantly between experimental systems .

How should OAT antibodies be stored and handled for optimal performance?

Proper storage and handling of OAT antibodies are critical for maintaining reactivity and specificity:

For lyophilized antibodies:

• Store at -20°C upon receipt .

• After reconstitution, store at 4°C for short-term use (one month) or aliquot and store at -20°C for long-term stability (six months) .

• Avoid repeated freeze-thaw cycles as they may compromise antibody performance .

For liquid formulations:

• Store at -20°C in buffer containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) .

• Aliquoting is recommended but not essential for -20°C storage .

• Maintain stable for one year when stored according to manufacturer recommendations .

Working dilutions should be prepared fresh before use, and appropriate controls should be included in each experiment to verify antibody performance .

How can I validate the specificity of an OAT antibody for my experimental system?

Validating antibody specificity is essential for reliable research outcomes. Comprehensive validation approaches include:

• Positive and negative controls: Include tissues or cell lines known to express OAT (e.g., K-562 cells, liver tissue) and those with minimal expression .

• Overexpression validation: Compare non-transfected and OAT-transfected cell lines (e.g., 293T cells) to confirm specificity. A distinct band at 48 kDa should be observed in transfected samples with significantly higher intensity .

• Molecular weight verification: Confirm detection at the predicted molecular weight (48 kDa for OAT) .

• Blocking peptide competition: Pre-incubate antibody with immunogenic peptide to demonstrate signal reduction in Western blot or immunostaining .

• Knockout/knockdown controls: If available, cells with CRISPR-Cas9 knockout or siRNA knockdown of OAT provide definitive validation of specificity .

• Cross-application validation: Confirm consistent results across multiple techniques (e.g., Western blot, IHC, IF) when appropriate .

Documentation of these validation steps significantly strengthens the reliability of research findings involving OAT antibodies .

How can OAT antibodies be used in cell surface biotinylation experiments?

Cell surface biotinylation using OAT antibodies provides valuable insights into protein trafficking and surface expression. The protocol involves:

• Cell preparation: Culture cells expressing OAT in appropriate medium and conditions until desired confluence is reached.

• Surface biotinylation: Incubate cells with membrane-impermeable NHS-SS-biotin (0.5 mg/ml in PBS) in two successive 20-minute incubations on ice with gentle shaking .

• Quenching: Rinse cells briefly with PBS/CM containing 100 mM glycine, then incubate with the same solution for 30 minutes on ice to quench unreacted biotin .

• Cell lysis: Lyse cells in buffer containing 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100 with protease inhibitor cocktail for 1 hour on ice .

• Isolation of biotinylated proteins: Clear lysates by centrifugation (16,000 g at 4°C) and add streptavidin-agarose beads to isolate membrane proteins .

• Detection: Perform Western blot analysis using anti-OAT antibody to detect surface expression levels .

This approach enables quantitative assessment of surface-expressed OAT under various experimental conditions, providing insights into protein trafficking mechanisms .

What methods can detect post-translational modifications of OAT using antibodies?

Investigating post-translational modifications (PTMs) of OAT provides crucial insights into its regulation and function. Key methodological approaches include:

• Ubiquitination detection:

  • Immunoprecipitate OAT using specific antibodies

  • Elute immunoprecipitated proteins with 1% SDS (37°C, 15 minutes)

  • Dilute SDS to 0.1% and incubate eluted proteins with streptavidin-agarose beads

  • Perform immunoblotting using anti-ubiquitin antibody (e.g., P4D1) to detect ubiquitinated OAT

• Phosphorylation analysis:

  • Use phospho-specific antibodies in Western blot following immunoprecipitation with OAT antibody

  • Alternatively, perform mass spectrometry analysis after immunoprecipitation to identify phosphorylation sites

• Other PTM investigations:

  • For glycosylation: Use enzymatic deglycosylation followed by Western blot to detect mobility shifts

  • For acetylation or methylation: Employ modification-specific antibodies after OAT immunoprecipitation

These approaches require thorough optimization and appropriate controls to ensure specificity and reliability of the results .

How can internalization assays be performed using OAT antibodies?

Investigating OAT internalization dynamics provides valuable insights into protein trafficking and regulation. A validated internalization assay protocol includes:

• Surface labeling: Biotinylate OAT-expressing cells with 0.5 mg/ml sulfo-NHS-SS-biotin at 4°C .

• Internalization initiation: Incubate biotinylated cells at 37°C in PBS containing either experimental treatment (e.g., 1 μM PMA) or vehicle for designated time periods .

• Surface biotin stripping: Remove remaining cell-surface biotin by incubating cells 3 times for 20 minutes with freshly prepared 50 mM MesNa in NT buffer (150 mM NaCl, 1 mM EDTA, 0.2% bovine serum albumin, 20 mM Tris, pH 8.6) .

• Cell lysis and protein isolation: Lyse cells in appropriate buffer containing protease inhibitors .

• Separation and detection: Isolate biotinylated (internalized) proteins and analyze by Western blot using OAT antibodies .

• Quantification: Measure the ratio of internalized to total initial surface OAT to calculate internalization rates .

This assay enables quantitative assessment of OAT trafficking under various experimental conditions, including response to signaling molecules or drug treatments .

How are OAT antibodies utilized in immunological disorder research?

OAT antibodies serve as important tools in investigating immunological disorders, particularly in celiac disease research where antibodies to oat prolamines (avenins) have clinical relevance:

• Celiac disease investigations: Studies have revealed that children with celiac disease on normal diets have significantly higher levels of antibodies to avenin (both IgG and IgA) compared to reference children (P < 0.001) . These findings highlight the immunogenic potential of oat proteins in susceptible individuals.

• Cross-reactivity studies: Absorption tests using avenin and gliadin have demonstrated that anti-avenin and anti-gliadin antibodies are only absorbed by their corresponding proteins, suggesting no cross-reactivity between wheat and oat prolamines . This contradicts earlier assumptions about shared epitopes.

• Methodological approach:

  • Crude avenin preparation through extraction with ethanol and salt solution

  • Three-step ELISA for detection of both IgA and IgG antibodies

  • Comparative analysis between patient and reference groups

  • Correlation analysis with gliadin antibodies

• Clinical applications: Monitoring anti-avenin antibodies during gluten-free diet interventions supplemented with oats provides valuable data on immunological responses .

These approaches demonstrate how antibody-based methodologies contribute to understanding immune responses in celiac disease and other immunological conditions .

What methods are effective for identifying immunogenic epitopes using OAT antibodies?

Identification of immunogenic epitopes is critical for understanding disease mechanisms and developing diagnostic tools. Effective methodological approaches include:

• Monoclonal antibody reactivity testing: Using characterized monoclonal antibodies (e.g., G12 moAb against the 33-mer peptide of α-2 gliadin) to screen for cross-reactive epitopes in oat proteins .

• Fractionation and characterization:

  • Separate protein fractions using SDS-PAGE

  • Perform western blot with dual staining techniques (antibody recognition followed by total protein staining with nigrosin)

  • Identify reactive proteins in specific molecular weight ranges (e.g., 25-37 kDa for major avenin bands)

• Peptide identification via mass spectrometry:

  • Extract and purify protein fractions

  • Perform Nano-LC-MS/MS analysis to identify specific peptide sequences

  • Correlate identified peptides with antibody reactivity

• Functional validation:

  • Test identified peptides for their ability to activate dendritic cells (DC) from patients compared to healthy controls

  • Measure T-cell proliferation and cytokine production in response to identified peptides

These approaches enable precise identification of immunogenic epitopes, facilitating both basic research and clinical applications in immunological disorders .

What are common issues encountered with OAT antibodies and their solutions?

Researchers frequently encounter several technical challenges when working with OAT antibodies. Here are evidence-based solutions to common problems:

• Weak or absent signal in Western blot:

  • Verify protein loading (25-30 μg recommended for whole cell extracts)

  • Ensure appropriate SDS-PAGE conditions (10% gels typically effective)

  • Optimize primary antibody dilution (1:1000-1:5000 range for detection of endogenous protein)

  • Increase exposure time or enhance detection reagent sensitivity

  • Consider alternative sample preparation methods to preserve protein integrity

• High background in immunostaining:

  • Implement more stringent blocking (5% BSA or 5-10% normal serum from secondary antibody species)

  • Optimize antibody dilution (start with 1:200-1:500 for IF/ICC)

  • Increase washing duration and frequency between antibody incubations

  • For IHC, optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

• Cross-reactivity with unrelated proteins:

  • Validate antibody specificity using overexpression or knockdown/knockout controls

  • Pre-absorb antibody with recombinant protein if possible

  • Use more stringent washing conditions

  • Consider testing alternative clones with different epitope recognition

• Variable results between experiments:

  • Standardize protein extraction and handling procedures

  • Prepare fresh working dilutions for each experiment

  • Maintain consistent incubation times and temperatures

  • Include positive control samples in each experiment for normalization

Systematic troubleshooting using these approaches can significantly improve experimental outcomes when working with OAT antibodies.

How can researchers optimize immunoprecipitation protocols for OAT?

Successful immunoprecipitation (IP) of OAT requires careful optimization of multiple parameters. Based on established protocols, consider the following methodological refinements:

• Antibody selection and amount:

  • Choose antibodies validated specifically for IP applications

  • Titrate antibody amount (typically 2-5 μg per 500 μg protein lysate)

  • For difficult samples, consider crosslinking antibody to beads to prevent heavy chain interference in subsequent Western blot analysis

• Lysis buffer optimization:

  • Use buffers containing 1% Triton X-100 with 0.1% SDS for efficient solubilization

  • Include protease inhibitor cocktail (1:100 dilution) to prevent degradation

  • For studying PTMs, add appropriate inhibitors (phosphatase, deubiquitinase, etc.)

  • Pre-clear lysates to reduce non-specific binding

• Binding conditions:

  • Perform antibody incubation overnight at 4°C with gentle rotation

  • Use protein A/G beads for rabbit IgG antibodies

  • Wash beads thoroughly (3-5 times) with decreasing salt concentrations

• Elution strategies:

  • For subsequent Western blot: Standard SDS sample buffer at 95°C

  • For mass spectrometry: Consider gentler elution with peptide competition or acidic glycine buffer

  • For studying interacting partners: 1% SDS at 37°C for 15 minutes

• Controls:

  • Include isotype control antibody IP

  • When possible, include samples with OAT knockdown/knockout

  • For transfected systems, compare with non-transfected controls

These methodological refinements significantly enhance the specificity and yield of OAT immunoprecipitation experiments, particularly for investigating protein interactions and post-translational modifications .

What are emerging applications and future directions for OAT antibody research?

The landscape of OAT antibody applications continues to evolve with advances in molecular biology techniques and disease research. Several promising directions are emerging:

• Single-cell analysis: Integration of OAT antibodies with single-cell technologies to investigate cellular heterogeneity in normal and pathological states.

• Multiplexed imaging: Combining OAT antibodies with other markers in multiplexed immunofluorescence or mass cytometry to understand complex cellular relationships and protein networks.

• Therapeutic targeting: Development of OAT-targeting therapeutic antibodies based on insights gained from research-grade antibodies, particularly in metabolic disorders.

• Biomarker development: Validation of OAT as a diagnostic or prognostic biomarker in various pathological conditions, utilizing antibody-based detection methods.

• Structural and functional studies: Epitope mapping and conformational analysis of OAT to better understand its regulation and function in health and disease.

• Improved detection methodologies: Development of more sensitive and specific detection systems for both research and clinical applications.