DHTKD1 Antibody

What is the optimal method for validating DHTKD1 antibody specificity in mitochondrial metabolism studies?

To ensure reliable results, researchers should implement a multi-step validation process:

• Western Blot Controls: Use DHTKD1 knockout (KO) cell lines (e.g., CRISPR-edited HAP-1 cells) as negative controls to confirm antibody specificity . Wild-type (WT) cells serve as positive controls.

• Immunoprecipitation (IP) Experiments: Validate antibody performance by co-IP with known interactors (e.g., OGDH, DLST, DLD) in mitochondrial lysates .

• Proximity Ligation Assay (PLA): Confirm spatial proximity between DHTKD1 and OGDH or DLST in fixed cells to assess interaction specificity .

• Functional Assays: Measure downstream metabolites (e.g., 2-aminoadipic acid) in KO vs. WT cells to correlate antibody detection with enzymatic activity .

Key Data from Validation Studies:

How do DHTKD1 antibodies perform in detecting mitochondrial complex interactions?

DHTKD1 antibodies are critical for studying hybrid complexes involving OGDH (2-oxoglutarate dehydrogenase) and DLST (dihydrolipoyl succinyltransferase). Key considerations include:

• Cross-reactivity: Anti-DHTKD1 antibodies may weakly bind OGDH due to ~38% amino acid homology. Validate specificity using OGDH-specific antibodies or KO cell lines .

• IP Efficiency: Optimize IP buffer conditions (e.g., 0.1% Triton X-100, 150 mM NaCl) to preserve mitochondrial membrane integrity and complex interactions .

• Application Limitations: Avoid using DHTKD1 antibodies for detecting non-mitochondrial isoforms or in species with divergent homology (e.g., zebrafish vs. human) .

Case Study: In HEK-293 cells, anti-DHTKD1 antibodies co-IP with OGDH and DLST, confirming hybrid complex formation. KO cells eliminate co-IP signals, validating specificity .

What experimental designs address conflicting data on DHTKD1’s role in glutaryl-CoA metabolism?

Conflicting results arise from partial redundancy with OGDH and substrate overlap. To resolve discrepancies:

• Dual KO Models: Generate Gcdh/Dhtkd1 double KO cells to assess glutaryl-CoA accumulation independent of OGDH activity .

• Metabolomic Profiling: Measure glutaric acid (GA), 3-hydroxyglutaric acid (3-HGA), and 2-aminoadipic acid (2-AAA) in KO vs. WT cells to distinguish pathway contributions .

• Enzymatic Assays: Quantify OADHc (2-oxoadipate dehydrogenase complex) and OGDHc activities in lysates to confirm substrate overlap .

Contradiction Resolution Example:

• Claim: DHTKD1 inhibition reduces glutaryl-CoA in GA1 .

• Counterclaim: Gcdh/Dhtkd1 double KO mice retain elevated C5DC (glutarylcarnitine), indicating residual OGDH-mediated flux .

• Solution: Use metabolomics to confirm whether GA/3-HGA (DHTKD1-dependent) or 2-AAA (OGDH-dependent) accumulates in specific models .

How do researchers optimize DHTKD1 antibody performance in mitochondrial respiration studies?

To study DHTKD1’s impact on OXPHOS (oxidative phosphorylation):

• Cell Model Selection: Use HAP-1 cells (haploid, human-derived) for clean KO phenotypes. Avoid cancer cell lines with variable mitochondrial function .

• Seahorse Assay Protocols:

  • Basal Respiration: Measure OCR (oxygen consumption rate) in WT vs. KO cells.

  • Stress Tests: Add oligomycin (ATP synthase inhibitor) and FCCP (protonophore) to assess proton leak and maximal respiration .

• Western Blot Normalization: Use Complex V (ATP5A) or COX IV as loading controls to account for mitochondrial content .

Mitochondrial Respiration Data:

What are advanced applications of DHTKD1 antibodies in metabolic disease research?

• Hybrid Complex Analysis: Use PLA to map DHTKD1-OGDH interactions in diabetic or neurodegenerative models .

• Metabolic Flux Studies: Track 13C-labeled lysine or glutamine through DHTKD1-dependent pathways using LC-MS/MS .

• GWAS Integration: Validate DHTKD1 SNPs associated with cardiometabolic traits using CRISPR-edited isogenic cell lines .

Example Protocol for PLA:

• Fixation: 4% PFA, 15 min at RT.

• Blocking: 5% BSA + 0.1% Triton X-100.

• Primary Antibodies: Mouse anti-DHTKD1 + rabbit anti-OGDH (1:200 dilution, overnight, 4°C).

• Secondary Probes: PLA probes with DNA-based detection (e.g., Duolink) .

How do researchers troubleshoot non-specific DHTKD1 antibody signals in complex lysates?

Common issues and solutions:

• Cross-reactivity with OGDH:

  • Test: Incubate lysates with excess OGDH peptide (blocking control).

  • Alternative: Use rabbit monoclonal antibodies with epitope mapping .

• Mitochondrial Matrix Contamination:

  • Solution: Pre-clear lysates with anti-TOM20 (mitochondrial membrane marker) IP to remove non-specific binding .

• Overexposure in WB:

  • Fix: Reduce primary antibody concentration (e.g., 1:500 → 1:1000) and exposure time .

Troubleshooting Workflow:

What role do DHTKD1 antibodies play in studying lysine degradation disorders?

In glutaric aciduria type 1 (GA1) and 2-aminoadipic aciduria (AMOXAD):

• Pathway Visualization: Track glutaryl-CoA and 2-oxoadipate flux via OADHc and OGDHc using antibody-based IP/MS .

• Therapeutic Target Validation: Assess substrate reduction efficacy by monitoring 2-AAA/OA accumulation in KO models .

• Biomarker Development: Quantify DHTKD1 protein levels in patient-derived cells to correlate with disease severity .

Clinical Relevance Data:

How do DHTKD1 antibodies inform mitochondrial protein complex studies?

• Hybrid Complex Formation: Co-IP with DLST and DLD to confirm OADHc/OGDHc interactions .

• Protein Stability: Use cycloheximide chase assays to assess DHTKD1 degradation in stress conditions (e.g., hypoxia) .

• Post-translational Modifications: Detect phosphorylation or ubiquitination sites via site-specific antibodies .

Case Study: In HEK-293 cells, DHTKD1 interacts with OGDH and DLST, forming a hybrid complex. This interaction is lost in DHTKD1 KO cells, confirming antibody specificity .

What are emerging trends in DHTKD1 antibody applications?

• Single-Cell Analysis: Combine DHTKD1 antibodies with mitochondrial markers (e.g., Tom20) for spatially resolved metabolomics .

• CRISPR Screens: Use DHTKD1 antibodies to validate gene knockouts in high-throughput screens for metabolic diseases .

• Proteome Profiling: Quantify DHTKD1 abundance across tissues using targeted mass spectrometry .

Future Directions: