DLAT Monoclonal Antibody

What is DLAT and why is it significant in biochemical research?

DLAT (dihydrolipoamide S-acetyltransferase) is a critical component of the pyruvate dehydrogenase complex, functioning as the E2 component (PDC-E2). With a calculated molecular weight of 70 kDa and comprising 647 amino acids, this mitochondrial protein plays an essential role in cellular energy metabolism by catalyzing the conversion of pyruvate to acetyl-CoA. The protein is encoded by the DLAT gene (Gene ID: 1737) and has been identified as a 70 kDa mitochondrial autoantigen in primary biliary cirrhosis. Understanding DLAT function contributes significantly to research on metabolic disorders and autoimmune conditions affecting mitochondrial function .

What are the key structural and biochemical characteristics of commercially available DLAT monoclonal antibodies?

DLAT monoclonal antibodies are typically mouse-derived IgG1 isotype antibodies that target specific epitopes on the human DLAT protein. Currently available antibodies are unconjugated, supplied in liquid form (1 mg/mL concentration), and purified using Protein G Magarose purification methods. These antibodies are commonly stored in PBS-only buffer (BSA and azide-free) to facilitate further conjugation for various applications. The antibodies are generated using recombinant DLAT fusion proteins as immunogens and are specifically designed to recognize both native and denatured forms of human DLAT with high specificity .

How do researchers distinguish between different epitope-targeting DLAT monoclonal antibodies?

Researchers can distinguish between different DLAT monoclonal antibodies based on the specific epitope regions they target. Epitope mapping is crucial for antibody characterization and involves identifying the precise amino acid sequence recognized by each antibody. Modern epitope-directed monoclonal antibody production methods utilize in silico-predicted epitopes (typically 13-24 residues long) to generate antibodies against spatially distant sites on the target protein. This approach allows researchers to select antibodies targeting different domains of DLAT, enabling validation schemes applicable to two-site ELISA, western blotting, and immunocytochemistry. The use of short antigenic peptides of known sequence facilitates direct epitope mapping, which is essential for comprehensive antibody characterization and determining the most appropriate applications for each antibody .

What validated applications are currently available for DLAT monoclonal antibodies in research settings?

DLAT monoclonal antibodies have been validated for multiple research applications including:

• Cytometric bead array: Particularly useful for quantitative multiplex protein detection with matched antibody pairs (e.g., 68303-4-PBS capture and 68303-5-PBS detection).

• Indirect ELISA: Validated for protein quantification in complex samples.

• Western blotting (WB): For detecting denatured DLAT protein in cell lysates from various cell lines including Jurkat, A549, U251, LNCAP, and HeLa.

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing subcellular localization of DLAT.

• Immunoprecipitation (IP): For isolating DLAT protein complexes from cellular samples.

The selection of application should be based on the specific research question, with optimization recommended for each experimental system .

What methodological considerations should researchers account for when designing experiments with DLAT antibodies?

When designing experiments using DLAT monoclonal antibodies, researchers should consider:

• Antibody format selection: Choose between unconjugated antibodies (requiring secondary detection) or directly conjugated antibodies based on experimental design.

• Matched pair compatibility: For two-site assays, select validated antibody pairs targeting different, non-competing epitopes.

• Species cross-reactivity: Most current DLAT antibodies are validated for human samples, with some showing rabbit reactivity. Confirm species compatibility before use.

• Storage and handling: Maintain antibody integrity by storing at -80°C and minimizing freeze-thaw cycles.

• Positive and negative controls: Include appropriate controls such as DLAT-overexpressing and knockdown cell lines.

• Buffer compatibility: Consider the PBS-only formulation (BSA and azide-free) when planning conjugation or specialized applications.

• Optimization: Titrate antibody concentrations for each application to achieve optimal signal-to-noise ratio .

How can researchers optimize DLAT antibody performance in multiplex immunoassays?

Optimizing DLAT antibody performance in multiplex immunoassays requires careful consideration of several factors:

• Antibody pair selection: Use validated matched pairs (e.g., 68303-8-PBS capture and 68303-9-PBS detection) specifically designed for multiplexing.

• Conjugation chemistry: For conjugation-ready antibodies in PBS-only buffer, select appropriate conjugation chemistry based on the detection platform.

• Cross-reactivity assessment: Test for potential cross-reactivity with other targets in the multiplex panel.

• Signal optimization: Titrate antibody concentrations to achieve comparable signal strength across all analytes.

• Buffer optimization: Adjust assay buffers to minimize background and maximize specific signal.

• Validation: Perform spike-recovery experiments with known concentrations of recombinant DLAT.

• Data analysis: Apply appropriate statistical methods for multiplex data interpretation.

Recent advances in ELISA assay miniaturization, facilitated by novel DEXT microplates, allow for rapid screening with concomitant epitope identification, making multiplex approaches with DLAT antibodies more accessible and efficient .

What are the most common sources of variability in DLAT antibody experiments, and how can they be mitigated?

Common sources of variability in DLAT antibody experiments include:

Performance inconsistencies and poor validation of antibodies contribute significantly to irreproducible and misleading data in research. To mitigate these issues, researchers should thoroughly validate antibodies for their specific application and experimental system before conducting critical experiments .

How can researchers validate the specificity of DLAT monoclonal antibodies to ensure experimental reliability?

To validate DLAT monoclonal antibody specificity, researchers should implement a comprehensive validation strategy:

• Western blot analysis: Confirm single band of expected molecular weight (70 kDa) in positive control samples with absence in negative controls.

• Knockout/knockdown validation: Compare antibody reactivity in DLAT-expressing versus DLAT-depleted samples.

• Epitope competition assay: Pre-incubate antibody with excess recombinant DLAT peptide to confirm specific blocking of signal.

• Cross-reactivity assessment: Test antibody against related family members or proteins with similar domains.

• Multiple antibody validation: Use antibodies targeting different DLAT epitopes and compare detection patterns.

• Orthogonal technique validation: Confirm DLAT detection using alternative methods (e.g., mass spectrometry).

• Recombinant expression: Test antibody against purified recombinant DLAT protein.

This multi-faceted approach helps ensure that experimental observations are truly related to DLAT detection rather than non-specific binding or cross-reactivity .

How can epitope-directed DLAT monoclonal antibodies be leveraged for investigating protein-protein interactions?

Epitope-directed DLAT monoclonal antibodies offer powerful tools for investigating protein-protein interactions through several methodological approaches:

• Co-immunoprecipitation (Co-IP): Antibodies targeting non-functional epitopes of DLAT can precipitate intact protein complexes, allowing identification of interaction partners by mass spectrometry or western blotting.

• Proximity ligation assay (PLA): Combining DLAT antibodies with antibodies against potential interaction partners enables visualization and quantification of protein interactions in situ with single-molecule sensitivity.

• FRET-based interaction studies: Conjugating DLAT antibodies with donor fluorophores and partner protein antibodies with acceptor fluorophores facilitates energy transfer measurements indicative of close molecular proximity.

• Competitive binding assays: Using epitope-mapped antibodies to block specific domains of DLAT can reveal which regions are essential for particular protein-protein interactions.

• Antibody inhibition studies: Introducing epitope-specific antibodies into cellular systems can disrupt specific interactions, revealing their functional significance.

By selecting antibodies targeting specific epitopes that don't interfere with interaction domains or by deliberately targeting interaction interfaces, researchers can gain detailed insights into DLAT's role in the pyruvate dehydrogenase complex and potentially discover novel interaction partners .

What methodological approaches can be used to investigate DLAT post-translational modifications using monoclonal antibodies?

Investigating DLAT post-translational modifications (PTMs) requires specialized methodological approaches:

• PTM-specific antibodies: Develop or obtain antibodies specifically recognizing modified forms of DLAT (e.g., phosphorylated, acetylated, lipoylated).

• Two-dimensional western blotting: Separate proteins by charge (reflecting PTM status) and molecular weight before probing with DLAT antibodies.

• Immunoprecipitation followed by mass spectrometry: Use DLAT antibodies to enrich the protein, then analyze PTMs by mass spectrometry.

• Sequential immunoprecipitation: First immunoprecipitate with general DLAT antibody, then with PTM-specific antibodies to determine modified fraction.

• In vitro modification assays: Treat recombinant DLAT with specific enzymes (kinases, acetyltransferases) and detect changes using general and PTM-specific antibodies.

• Cellular perturbation studies: Manipulate cellular signaling pathways and assess changes in DLAT PTM status.

• Site-directed mutagenesis coupled with antibody detection: Mutate potential modification sites and assess changes in antibody recognition patterns.

These approaches enable researchers to map the PTM landscape of DLAT and understand how these modifications regulate its function within the pyruvate dehydrogenase complex .

How can researchers address potential immunogenicity issues when using DLAT monoclonal antibodies in translational research?

Addressing immunogenicity concerns in translational research involving DLAT monoclonal antibodies requires careful consideration of several factors:

• Antibody humanization: If considering therapeutic applications, mouse monoclonal antibodies should undergo humanization to reduce immunogenicity.

• Anti-drug antibody (ADA) monitoring: Develop and validate assays to detect antibodies generated against the administered DLAT monoclonal antibody.

• HLA haplotype analysis: Consider HLA haplotypes as potential biomarkers to predict vulnerability to ADA formation, as different patients may exhibit different immunogenic responses.

• Immunogenicity prediction algorithms: Utilize computational tools to identify potential T-cell epitopes that might trigger immune responses.

• Co-administration strategies: Consider immunosuppressors that may attenuate ADA responses (similar to methotrexate's effect with TNFα antagonistic antibodies).

• Dosing regimen optimization: Design dosing schedules that minimize immunogenicity while maintaining efficacy.

• Formulation optimization: Adjust antibody formulation to reduce aggregation and other physical characteristics that enhance immunogenicity.

Understanding that over 90% of therapeutic proteins cause some degree of immunogenicity with production of ADAs, researchers must implement strategies to mitigate this response, particularly if DLAT antibodies are being considered for diagnostic or therapeutic applications .

How are advances in epitope-directed monoclonal antibody production improving research applications for DLAT?

Recent advances in epitope-directed monoclonal antibody production are revolutionizing DLAT research capabilities:

• In silico epitope prediction: Computational methods now enable precise identification of antigenic regions on DLAT, allowing for targeted antibody development against specific functional domains.

• Multi-epitope targeting: Within a single hybridoma production cycle, antibodies against multiple in silico-predicted epitopes on DLAT can be generated, providing comprehensive protein coverage.

• Optimized antigen presentation: Short antigenic peptides (13-24 residues) presented as three-copy inserts on surface-exposed loops of thioredoxin carriers produce high-affinity monoclonal antibodies reactive to both native and denatured DLAT.

• Miniaturized screening technologies: Novel DEXT microplates enable rapid hybridoma screening with simultaneous epitope identification, accelerating the development process.

• Spatial epitope mapping: Generating antibodies against spatially distant sites on DLAT facilitates validation schemes for two-site ELISA, western blotting, and immunocytochemistry.

• Enhanced characterization: Direct epitope mapping using known peptide sequences provides crucial information for antibody characterization and application optimization.

These methodological improvements address critical issues of antibody quality, validation, and utility, ultimately leading to more reproducible and reliable research outcomes with DLAT antibodies .

What considerations should researchers make when selecting between different DLAT monoclonal antibody clones for specific applications?

When selecting between different DLAT monoclonal antibody clones, researchers should consider:

• Epitope location: Choose antibodies targeting epitopes relevant to the research question (e.g., functional domains, interaction sites, or PTM locations).

• Application validation: Verify that the antibody has been validated specifically for your intended application (WB, ICC, IP, ELISA).

• Sample compatibility: Ensure the antibody works with your sample type (cell lysates, tissue sections, fixed cells).

• Native vs. denatured recognition: Some antibodies recognize only denatured DLAT (useful for Western blot) while others recognize native conformation (better for IP or ICC).

• Clone specificity profile: Review cross-reactivity data and specificity validation experiments for each clone.

• Matched pair compatibility: For two-site assays, select antibodies validated to work together without epitope competition.

• Technical support availability: Consider whether comprehensive protocols and troubleshooting guidance are available.

• Lot-to-lot consistency: Review quality control data demonstrating reproducibility between production lots.

Careful antibody selection based on these criteria helps ensure experimental success and reliable results, addressing the widespread concerns about antibody quality and validation in the research community .

What standardized reporting practices should researchers follow when publishing results obtained using DLAT monoclonal antibodies?

To ensure reproducibility and transparency when publishing results obtained using DLAT monoclonal antibodies, researchers should adhere to these standardized reporting practices:

• Complete antibody identification: Report manufacturer, catalog number, clone ID, lot number, and RRID (Research Resource Identifier).

• Validation documentation: Include or reference validation data demonstrating antibody specificity for DLAT, ideally including positive and negative controls.

• Detailed methods: Provide complete protocols including antibody concentration, incubation conditions, buffer compositions, and detection methods.

• Control experiments: Clearly describe all controls used to validate findings, including isotype controls and knockdown/knockout validations.

• Image acquisition parameters: For microscopy applications, report all relevant parameters (exposure times, gain settings, post-processing).

• Quantification methods: Detail the methods used for signal quantification and statistical analysis.

• Raw data availability: Consider providing access to unprocessed images or data in supplementary materials or repositories.

• Reproducibility assessment: Report the number of technical and biological replicates and inter-experimental variability.

These reporting practices address the persistent issues of irreproducibility in antibody-based research and help establish a more robust foundation for DLAT-related discoveries .

How can researchers accurately interpret DLAT antibody signals in complex biological samples with potential interfering factors?

Accurate interpretation of DLAT antibody signals in complex biological samples requires methodical approach to identifying and controlling for potential interfering factors:

• Appropriate controls:

  • Positive controls: Samples with confirmed DLAT expression

  • Negative controls: DLAT-knockout samples or immunodepleted samples

  • Isotype controls: Non-specific antibodies of same isotype to assess background

• Signal validation strategies:

  • Peptide competition: Pre-incubation with specific DLAT peptide should abolish specific signal

  • Multiple antibodies: Confirm signals using antibodies targeting different DLAT epitopes

  • Orthogonal techniques: Validate findings using non-antibody methods (e.g., mass spectrometry)

• Interference mitigation:

  • Sample pre-clearing: Remove non-specific binding proteins

  • Blocking optimization: Test different blocking reagents to reduce background

  • Buffer optimization: Adjust salt and detergent concentrations to enhance specificity

• Quantitative considerations:

  • Standard curves: Use recombinant DLAT for quantitative applications

  • Signal linearity: Verify linear relationship between DLAT concentration and signal

  • Signal-to-noise ratio: Calculate and report as quality metric

These approaches help differentiate true DLAT signals from artifacts, addressing the quality concerns that have contributed to irreproducible and misleading data in antibody-based research .