Dlat Antibody

What is DLAT and why is it important in cellular metabolism?

DLAT (dihydrolipoamide S-acetyltransferase, also known as PDC-E2) is a key enzyme in the pyruvate dehydrogenase complex that catalyzes the conversion of pyruvate to acetyl-CoA in the mitochondria. This conversion represents a critical step in cellular energy metabolism, linking glycolysis to the citric acid cycle. DLAT dysregulation has been associated with several metabolic diseases including diabetes and cancer, highlighting its significance in cellular energetics and potential as a therapeutic target . The protein has a molecular weight of approximately 69-70 kDa and is primarily localized in the mitochondrial matrix, making it an important marker for mitochondrial function studies .

What applications are most suitable for DLAT antibody-based experiments?

DLAT antibodies have been validated for multiple applications with varying optimal dilutions:

The application should be selected based on your specific research question. For protein expression quantification, WB is most appropriate, while cellular localization studies benefit from IF/ICC approaches .

How should I optimize DLAT antibody conditions for Western blot applications?

Optimization of DLAT antibody conditions for Western blot requires a systematic approach:

• Initial titration: Start with manufacturer's recommended dilution (typically 1:1000-1:5000 for most commercial DLAT antibodies)

• Sample preparation: Ensure proper cell lysis with protease inhibitors to prevent degradation

• Loading controls: Include mitochondrial markers (e.g., VDAC) alongside standard loading controls

• Blocking optimization: Test both BSA and milk-based blocking solutions (5% concentration) as performance may vary

• Incubation conditions: Compare overnight incubation at 4°C versus 1-2 hours at room temperature

• Detection system selection: HRP-conjugated secondary antibodies with ECL detection systems typically provide optimal results for DLAT visualization

When troubleshooting weak signals, increasing antibody concentration, extending incubation time, or switching to more sensitive detection reagents may improve results . Most researchers report consistent detection of DLAT at 69-70 kDa across human, mouse, and rat samples .

What are the critical considerations for immunohistochemistry experiments with DLAT antibodies?

Successful IHC experiments with DLAT antibodies require attention to several critical factors:

• Antigen retrieval method: TE buffer (pH 9.0) is frequently recommended, though citrate buffer (pH 6.0) serves as an alternative when needed

• Tissue fixation impact: Overfixation can mask epitopes; optimize fixation time (typically 24-48 hours in 10% formalin)

• Antibody selection: Rabbit polyclonal antibodies often provide superior sensitivity for DLAT detection in tissue sections

• Controls: Include both positive controls (tissues with known DLAT expression like liver) and negative controls (antibody diluent only)

• Signal amplification: Consider using polymer-based detection systems for enhanced sensitivity

• Counterstaining optimization: Light hematoxylin counterstaining preserves DLAT signal visibility

Researchers have successfully detected DLAT in human liver cancer, colon cancer, and stomach cancer tissues, as well as in mouse kidney tissue samples . The mitochondrial localization pattern should appear as cytoplasmic punctate staining in positive cells.

How can DLAT antibodies be utilized to investigate mitochondrial dysfunction in metabolic diseases?

DLAT antibodies serve as powerful tools for investigating mitochondrial dysfunction through multiple methodological approaches:

• Differential expression analysis: Compare DLAT levels in diseased versus healthy tissues using quantitative Western blot analysis normalized to mitochondrial mass markers

• Post-translational modification studies: Use phospho-specific DLAT antibodies to assess regulatory modifications that affect enzyme activity

• Protein-protein interaction networks: Employ co-IP with DLAT antibodies followed by mass spectrometry to identify altered interaction partners in disease states

• Mitochondrial morphology correlation: Combine DLAT immunostaining with mitochondrial network visualization (using MitoTracker dyes) to correlate enzyme distribution with structural abnormalities

• Functional assays: Integrate oxygen consumption measurements with DLAT expression analysis to establish direct links between protein levels and respiratory capacity

Research has shown altered DLAT expression or modification in diabetes and various cancers . When designing these studies, careful selection of appropriate cellular models that recapitulate disease-specific metabolic alterations is essential for meaningful results.

What are the implications of anti-DLAT autoantibodies in immune-mediated neuropathies?

Recent research has identified significant associations between anti-DLAT autoantibodies and immune-mediated neuropathies:

• Prevalence patterns: Anti-DLAT antibodies were detected in 18% (29/160) of patients with chronic inflammatory demyelinating polyneuropathy (CIDP), 10% (6/58) of patients with sensory neuropathy, and 4% (2/47) of patients with multiple sclerosis

• Clinical correlations: CIDP patients with anti-DLAT antibodies showed higher rates of:

  • Sensory ataxia (69% vs. 37% in antibody-negative cases)

  • Cranial nerve disorders (24% vs. 9%)

  • Malignancy comorbidity (20% vs. 5%)

• Pathophysiological mechanisms: DLAT is highly expressed in dorsal root ganglion cells, suggesting a mechanistic link to the sensory-predominant phenotype observed in affected patients

• Diagnostic application: Anti-DLAT antibodies may serve as biomarkers specifically for sensory-dominant neuropathies

• Cytotoxicity potential: Ex vivo studies using dorsal root ganglion neurons have been conducted to assess the direct pathogenic effects of these autoantibodies on neuronal viability and axonal outgrowth

This emerging research suggests that DLAT antibody testing could provide valuable diagnostic information for patients with predominantly sensory neuropathic presentations, potentially guiding treatment approaches and prognosis assessment.

How can I validate the specificity of my DLAT antibody for research applications?

Comprehensive validation of DLAT antibody specificity requires multiple complementary approaches:

• Genetic validation:

  • DLAT knockdown/knockout controls using siRNA, shRNA, or CRISPR-Cas9 technology

  • Comparison of signal reduction across multiple applications (WB, IF, IHC)

• Biochemical validation:

  • Pre-absorption with recombinant DLAT protein

  • Peptide competition assays using the immunizing peptide/sequence

  • Testing across multiple species to confirm cross-reactivity claims

• Orthogonal validation:

  • Comparison with multiple independent antibodies targeting different DLAT epitopes

  • Correlation with mRNA expression data

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Application-specific controls:

  • WB: Molecular weight verification (69-70 kDa) and single-band specificity

  • IF/IHC: Co-localization with established mitochondrial markers

  • IP: Mass spectrometry validation of pulled-down proteins

Research publications have employed combinations of these approaches to establish DLAT antibody specificity, with genetic depletion methods showing particularly convincing validation outcomes .

What are the recommended procedures for optimizing immunofluorescence experiments with DLAT antibodies?

Optimizing immunofluorescence experiments for DLAT detection requires careful attention to the protein's mitochondrial localization:

• Fixation optimization:

  • Compare 4% paraformaldehyde (10-15 minutes) versus methanol fixation (-20°C, 10 minutes)

  • Evaluate permeabilization agents (0.1-0.5% Triton X-100, 0.1% saponin) for optimal mitochondrial access

• Antibody parameters:

  • Titrate antibody dilutions (1:50-1:500 range typical for primary incubation)

  • Test incubation times and temperatures (1 hour at room temperature versus overnight at 4°C)

• Co-localization strategy:

  • Include established mitochondrial markers (MitoTracker, TOM20, VDAC)

  • Use confocal microscopy with Z-stack acquisition for accurate spatial relationship assessment

• Signal optimization:

  • Compare signal amplification methods (tyramide signal amplification versus standard fluorophore-conjugated secondaries)

  • Evaluate nuclear counterstains that preserve mitochondrial signal (DAPI or Hoechst at optimized concentrations)

• Controls:

  • Include IgG isotype controls at equivalent concentrations

  • Prepare secondary-only controls to assess background

HepG2 cells consistently show strong DLAT expression and represent an excellent positive control for protocol optimization .

How can DLAT antibodies be employed in investigating the role of PDC dysregulation in neurodegenerative disorders?

DLAT antibodies offer multiple methodological approaches for investigating pyruvate dehydrogenase complex (PDC) dysregulation in neurodegenerative contexts:

• Regional expression analysis:

  • Use IHC with DLAT antibodies to map expression patterns across brain regions

  • Quantify expression changes in affected versus spared regions in post-mortem tissue

• Cellular energy metabolism profiling:

  • Combine DLAT immunostaining with functional assays (ATP production, lactate/pyruvate ratios)

  • Correlate DLAT levels with mitochondrial membrane potential assessments

• Oxidative stress relationships:

  • Dual labeling with DLAT and oxidative stress markers (4-HNE, 3-nitrotyrosine)

  • Assess post-translational modifications of DLAT under oxidative conditions

• Animal model validation:

  • Track longitudinal changes in DLAT expression in neurodegenerative disease models

  • Correlate with disease progression markers and behavioral outcomes

• Human biofluid applications:

  • Evaluate DLAT release into CSF as a potential biomarker

  • Assess anti-DLAT autoantibody presence in patient cohorts

Recent research has demonstrated high DLAT expression in neurons, particularly in the dorsal root ganglia, suggesting potential vulnerability of these cells to PDC dysfunction . This approach could provide mechanistic insights into energy failure hypotheses of neurodegeneration.

What experimental approaches can determine if anti-DLAT antibodies have direct pathogenic effects in neuropathies?

Determining the pathogenic potential of anti-DLAT antibodies requires multiple complementary experimental approaches:

• Ex vivo neurotoxicity assays:

  • Primary dorsal root ganglion neuron cultures exposed to patient-derived anti-DLAT antibodies

  • Quantification of:

    • Cell viability (LDH release, WST-8 assays)

    • Neurite outgrowth (β3-tubulin immunostaining)

    • Mitochondrial function (membrane potential, ROS production)

• Passive transfer models:

  • Injection of purified anti-DLAT antibodies into experimental animals

  • Assessment of:

    • Electrophysiological changes (nerve conduction studies)

    • Behavioral phenotypes (sensory testing, coordination)

    • Histopathological alterations

• Mechanism investigation:

  • Internalization studies of anti-DLAT antibodies into neurons

  • Analysis of PDC enzyme activity in the presence of antibodies

  • Evaluation of mitochondrial dynamics and morphology

• Human validation:

  • Correlation between antibody titers and disease severity

  • Longitudinal assessment of antibody levels and clinical outcomes

  • Response to treatments targeting antibody production/removal

Preliminary research has employed LDH and WST-8 assays with mouse DRG neurons to evaluate cytotoxicity of anti-DLAT antibody-containing patient sera, though comprehensive understanding requires integration of multiple methodological approaches .

What are the most common technical challenges when working with DLAT antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with DLAT antibodies that can be systematically addressed:

When troubleshooting DLAT antibody experiments, it's advisable to include multiple positive controls (HeLa, HepG2, and MCF-7 cells) that consistently show DLAT expression across different studies .

How should researchers interpret contradictory results between different DLAT antibody detection methods?

When faced with contradictory results between different detection methods, researchers should implement a systematic analytical approach:

• Epitope consideration:

  • Compare binding sites of different antibodies (N-terminal vs. C-terminal vs. internal domains)

  • Evaluate potential epitope masking in different applications (fixed vs. native conditions)

• Protocol compatibility analysis:

  • Assess whether fixation/preparation methods preserve the specific epitope

  • Consider whether denaturation (as in WB) versus native conformation (as in IP) affects recognition

• Cross-validation strategy:

  • Implement orthogonal detection methods (e.g., mass spectrometry)

  • Use genetic approaches (siRNA knockdown) to confirm specificity across methods

• Sample-specific factors:

  • Evaluate post-translational modifications that might differ between sample types

  • Consider splice variants or proteolytic processing in different tissues/cell lines

• Quantitative comparison:

  • Normalize signals relative to appropriate controls for each method

  • Conduct statistical analysis across multiple biological replicates

Research has shown that DLAT antibodies generally perform consistently across WB applications, while IHC and IF applications may show greater variability based on tissue preparation methods and epitope accessibility .

How are DLAT antibodies being used to investigate metabolic reprogramming in cancer cells?

DLAT antibodies are enabling multiple novel approaches to understanding cancer metabolic reprogramming:

• Expression profiling across cancer types:

  • Systematic IHC analysis of DLAT across tumor microarrays

  • Correlation with metabolic signatures and patient outcomes

  • Identification of cancer subtypes with PDC dysregulation

• Metabolic flux investigation:

  • Integration of DLAT protein levels with isotope tracing experiments

  • Assessment of pyruvate flux into TCA cycle versus lactate production

  • Correlation with mitochondrial respiration capacity

• Therapeutic response prediction:

  • Monitoring DLAT levels pre/post-treatment with metabolism-targeting drugs

  • Correlation with sensitivity to specific metabolic inhibitors

  • Development of companion diagnostic approaches

• Protein interaction landscapes:

  • Mapping altered DLAT protein complexes in malignant versus normal cells

  • Identification of cancer-specific regulatory mechanisms

  • Exploration of potential druggable interactions

• Post-translational modification analysis:

  • Assessment of phosphorylation, acetylation, or other modifications

  • Connection to oncogenic signaling pathways

  • Identification of regulatory mechanisms specific to cancer cells

DLAT expression has been successfully detected in multiple cancer cell lines, including HeLa, HepG2, MCF-7, and LNCaP, providing valuable model systems for these investigations .

What are the methodological considerations for using DLAT antibodies in neurodegenerative disease research?

Using DLAT antibodies effectively in neurodegenerative research requires specialized methodological adaptations:

• Tissue preparation optimization:

  • Short post-mortem intervals (<12 hours) preserve mitochondrial antigenicity

  • Controlled fixation protocols (4% PFA, 24-48 hours) maintain epitope recognition

  • Specialized antigen retrieval methods for neural tissues

• Cell type-specific analysis:

  • Double immunolabeling with neuronal, astrocytic, and microglial markers

  • Laser capture microdissection combined with Western blotting

  • Single-cell analysis approaches for heterogeneous neural populations

• Disease-specific considerations:

  • Age-matched controls to account for age-related mitochondrial changes

  • Region-specific sampling based on disease pathology pattern

  • Correlation with disease-specific markers (e.g., Aβ, tau, α-synuclein)

• Technical adaptations:

  • Autofluorescence quenching for neural tissue immunofluorescence

  • Modified extraction buffers for lipid-rich brain tissue

  • Extended incubation times for antibody penetration in tissue sections

• Validation approaches:

  • Comparison with functional mitochondrial assays

  • Correlation with transcriptomic data from same regions

  • Multimodal imaging combining antibody detection with metabolic measures

Research has demonstrated high DLAT expression in dorsal root ganglion cells, suggesting potential relevance to sensory neuron function and vulnerability in neurodegenerative conditions .

What role are DLAT antibodies playing in autoimmune disorder diagnostics and how might this evolve?

DLAT antibodies are emerging as important diagnostic tools for specific autoimmune disorders with evolving applications:

• Current diagnostic landscape:

  • Anti-DLAT antibodies detected in 18% of CIDP patients versus 0% of healthy controls

  • Especially prevalent in sensory-predominant neuropathies (69% with comorbid sensory ataxia)

  • Associated with distinct clinical phenotypes including cranial nerve involvement (24%)

• Assay methodologies:

  • Cell-based assays using DLAT-transfected cells show improved sensitivity

  • Tissue-based immunohistochemistry on dorsal root ganglia provides anatomical context

  • ELISA and Western blot validation confirm specificity

• Emerging clinical applications:

  • Differential diagnosis between inflammatory and hereditary neuropathies

  • Identification of potentially immunotherapy-responsive patient subgroups

  • Risk stratification for associated malignancies (20% comorbidity rate)

• Future diagnostic directions:

  • Development of standardized commercial assay platforms

  • Integration into comprehensive autoantibody panels for neuropathy workup

  • Longitudinal monitoring of antibody titers to assess treatment response

• Therapeutic implications:

  • Targeted immunotherapies for antibody-positive patients

  • Monitoring antibody levels to guide treatment duration

  • Preventive screening in high-risk patient populations

The recent identification of DLAT as an autoantigen in immune-mediated neuropathies represents an important advance in neurological diagnostics, with potential to improve patient classification and treatment selection .

How can advanced imaging techniques enhance DLAT antibody applications in mitochondrial research?

Advanced imaging technologies are revolutionizing DLAT antibody applications in mitochondrial research:

• Super-resolution microscopy approaches:

  • STED and STORM imaging to resolve DLAT distribution within mitochondrial subcompartments

  • Single-molecule localization microscopy to quantify DLAT clustering and organizational patterns

  • Correlative light-electron microscopy to connect protein localization with ultrastructural features

• Live-cell imaging innovations:

  • Development of cell-permeable DLAT-targeting nanobodies

  • FRET-based sensors to monitor DLAT interactions in real-time

  • Optogenetic approaches to manipulate DLAT function with spatial precision

• Multiplexed imaging strategies:

  • Cyclic immunofluorescence to assess DLAT in context of multiple mitochondrial proteins

  • Mass cytometry imaging to quantify DLAT alongside dozens of other targets

  • Spatial transcriptomics integration to correlate protein levels with gene expression

• Functional imaging correlations:

  • Combined DLAT immunofluorescence with mitochondrial potential dyes

  • Integration with metabolic sensors (ATP, NADH, pH)

  • Multiparametric imaging of mitochondrial dynamics and DLAT distribution

• Clinical translation approaches:

  • Development of PET tracers targeting DLAT or PDC activity

  • Ex vivo imaging of patient-derived tissues for personalized diagnostics

  • Correlative imaging with clinical neuroimaging (MRI, PET)

These advanced imaging approaches enable researchers to move beyond simple detection of DLAT and toward understanding its dynamic behavior, interactions, and functional states within the complex mitochondrial environment.

What are the current consensus recommendations for DLAT antibody validation in research?

The scientific community has developed evolving standards for DLAT antibody validation that reflect broader antibody validation principles:

• Multi-application validation:

  • Demonstration of consistent results across at least two independent methods (e.g., WB plus IHC)

  • Documentation of expected subcellular localization pattern (mitochondrial matrix)

  • Verification of molecular weight specificity (69-70 kDa band in WB)

• Genetic validation requirements:

  • Knockdown/knockout controls showing signal reduction

  • Rescue experiments with exogenous DLAT expression

  • Correlation with mRNA expression data

• Independent antibody confirmation:

  • Verification using antibodies targeting different DLAT epitopes

  • Comparison between monoclonal and polyclonal antibodies

  • Cross-validation between commercial sources

• Application-specific standards:

  • WB: Single band at expected molecular weight, reduced signal with knockdown

  • IHC/IF: Mitochondrial pattern, abolished with blocking peptide, reduced with knockdown

  • IP: Mass spectrometry confirmation of pulled-down proteins

• Reporting requirements:

  • Complete documentation of validation experiments in publications

  • Disclosure of catalog numbers, dilutions, and specific protocols

  • Availability of raw validation data

Following these validation standards ensures reliability and reproducibility of DLAT antibody-based research findings, particularly important given DLAT's emerging role in both basic metabolic research and clinical applications .

How should researchers select the optimal DLAT antibody for their specific experimental needs?

Selecting the optimal DLAT antibody requires systematic evaluation of multiple parameters aligned with specific experimental goals:

• Application-specific performance assessment:

  • Western blot: Prefer antibodies showing single band specificity at 69-70 kDa

  • IHC: Select antibodies validated on relevant tissue/species with appropriate controls

  • IF: Choose antibodies demonstrating clear mitochondrial localization pattern

  • IP: Prioritize antibodies with documented success in native protein binding

• Isotype and format considerations:

  • Polyclonal antibodies: Often provide higher sensitivity but potentially lower specificity

  • Monoclonal antibodies: Offer greater consistency between lots but may be epitope-restricted

  • Recombinant antibodies: Provide highest batch-to-batch reproducibility

• Epitope evaluation:

  • N-terminal vs. C-terminal targeting: Consider potential protein processing or interactions

  • Species conservation: Assess epitope sequence homology across target species

  • Post-translational modifications: Evaluate whether epitope contains modification sites

• Validation strength assessment:

  • Genetic validation (knockdown/knockout data)

  • Independent validation by multiple research groups

  • Publication record in high-quality peer-reviewed journals

• Technical specifications matching:

  • Reactivity with target species (human, mouse, rat)

  • Validated dilution ranges appropriate for application

  • Buffer compatibility with experimental conditions

When available, recombinant rabbit monoclonal antibodies targeting conserved DLAT regions often provide optimal performance across applications, as evidenced by consistent results in Western blot, IHC, and IF applications .