NDUFC2 Antibody

What is the role of NDUFC2 in mitochondrial function?

NDUFC2 functions as an essential subunit of the mitochondrial respiratory chain complex I. Research has demonstrated that NDUFC2 plays a crucial role in the assembly of the membrane arm of complex I, particularly involving the ND2 module and possibly the ND1 module. This protein is located in the intermembrane space and interacts with at least 12 other subunits across multiple modules, including NDUFA8 within the ND1 module, ND2 module subunits (NDUFA10, NDUFA11, NDUFS5, NDUFC1, and ND2), and ND4 module constituents (NDUFB1, NDUFB5, NDUFB10, NDUFB11, and ND4) . These interactions appear critical for both complex I assembly and stability. Complexome profiling has confirmed that loss of NDUFC2 causes concurrent loss of its associating subunits in the ND1 and ND2 modules, suggesting it may serve as a scaffold for ND1 module formation .

What methodological approaches are recommended for assessing NDUFC2 expression levels?

For optimal assessment of NDUFC2 expression levels, researchers should employ a multi-modal approach:

• RT-PCR Analysis: Extract total RNA (2μg recommended) and perform cDNA synthesis using random examer primers. For RT-PCR, use a 2× SYBR Green PCR Master Mix with the following conditions: initial denaturation at 94°C for 10 minutes followed by 40 cycles of 94°C for 15 seconds and 60°C for 15 seconds. Calculate mRNA concentrations using serially diluted standard curves and normalize to β-actin mRNA levels .

• Western Blot Analysis: For protein detection, use anti-NDUFC2 rabbit polyclonal antibody (recommended dilution 1:200) after standard SDS-PAGE and membrane transfer protocols. Visualization should be performed using appropriate HRP-conjugated secondary antibodies and chemiluminescence detection systems .

When comparing expression levels between experimental conditions, ensure consistent normalization strategies across all samples.

How can researchers verify NDUFC2 antibody specificity for experimental applications?

To verify NDUFC2 antibody specificity, implement these methodological controls:

• Positive and Negative Control Tissues/Cells: Include samples with known high NDUFC2 expression (e.g., mitochondria-rich tissues) alongside samples with experimentally reduced expression (NDUFC2-silenced cells) in immunoblotting experiments.

• Antibody Validation via Gene Silencing: Compare antibody reactivity patterns between wild-type cells and those subjected to NDUFC2 silencing via siRNA or shRNA approaches. The pattern observed in search results shows that specific NDUFC2 signals should decrease substantially in silenced cells .

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide prior to application in immunodetection to confirm binding specificity.

• Cross-Validation with Multiple Antibodies: When possible, use antibodies targeting different epitopes of NDUFC2 to confirm consistent detection patterns.

How can NDUFC2 antibodies be utilized in complexome profiling studies?

Complexome profiling represents a sophisticated approach for studying NDUFC2's role in complex I assembly. To implement this technique:

• Sample Preparation: Isolate intact mitochondria from relevant tissues or cell lines and solubilize using mild detergents (digitonin is recommended) to preserve protein-protein interactions.

• Blue Native PAGE (BN-PAGE): Separate protein complexes on gradient gels (3-12% or 4-16%), then cut the gel lane into equal slices for subsequent mass spectrometry analysis.

• Antibody Verification: Use NDUFC2 antibodies in parallel Western blot analyses of identical BN-PAGE samples to correlate mass spectrometry identification with immunodetection.

• Data Analysis: Track the migration patterns of NDUFC2 and other complex I subunits across the molecular weight range to identify assembly intermediates.

The search results indicate that complexome profiling of NDUFC2-deficient samples reveals specific aberrant complex I assembly intermediates consistent with stalling of complex I assembly, particularly affecting the ND2 module . This approach has proven valuable for elucidating the molecular mechanisms underlying complex I assembly and stability in cases of NDUFC2 dysfunction.

What experimental methods can differentiate between assembly defects and stability issues when studying NDUFC2 mutations?

Distinguishing between assembly defects and stability issues requires a multi-faceted experimental approach:

• Pulse-Chase Analysis: Label newly synthesized proteins with radioisotopes or other trackers, then monitor complex I formation over time in systems with wild-type versus mutant NDUFC2.

• Rescue Experiments: Introduce wild-type NDUFC2 using controlled expression systems (such as doxycycline-inducible lentiviral vectors) and monitor complex I assembly. The search results show that lentiviral transduction of wild-type NDUFC2 into cells with NDUFC2 mutations ameliorates complex I assembly defects, with increased levels of other complex I subunits (NDUFB8, NDUFA9, and NDUFV1) and greater assembly of the holoenzyme .

• Time-Course Analysis: Perform complexome profiling at multiple time points following induction of wild-type NDUFC2 expression to track the dynamics of complex I assembly.

• Inhibitor Studies: Apply proteasome inhibitors to distinguish between assembly failure and enhanced degradation of unstable complexes.

The search results suggest that NDUFC2 mutations can cause assembling complex I to stall at specific intermediates, leading to the accumulation of abnormal subcomplexes rather than merely accelerating degradation of fully formed complexes .

What are the key considerations when using NDUFC2 antibodies for analyzing patient samples with suspected mitochondrial disease?

When analyzing patient samples with suspected mitochondrial disease:

• Sample Preservation: Maintain strict protocols for sample collection and preservation to prevent artifactual loss of mitochondrial proteins.

• Control Selection: Include age-matched control samples processed under identical conditions.

• Mutation-Specific Effects: Consider that different mutations in NDUFC2 may affect antibody epitopes differently. For example, the search results describe two distinct homozygous variants (c.346_*7del and c.173A>T p.His58Leu) that both caused Leigh syndrome but might interact differently with antibodies depending on the targeted epitope .

• Complementary Assays: Always correlate antibody-based detection with functional assays of complex I activity and assembly.

• Tissue Specificity: Be aware that expression patterns and consequences of NDUFC2 mutations may vary between tissues, potentially affecting antibody reactivity patterns.

How can researchers effectively measure the impact of NDUFC2 dysfunction on mitochondrial activity?

To comprehensively assess the impact of NDUFC2 dysfunction on mitochondrial activity:

Research has demonstrated that NDUFC2 disruption alters complex I assembly and activity, reduces mitochondrial membrane potential and ATP levels, and increases reactive oxygen species production .

What strategies can be employed to investigate the relationship between NDUFC2 dysfunction and inflammatory pathways?

To investigate NDUFC2 dysfunction's impact on inflammatory pathways:

• Inflammatory Gene Expression Profiling: Utilize targeted arrays such as the RT² Profiler PCR array for inflammation markers. The search results mention using the Rat Inflammation array (SuperArray Bioscience) to compare gene expression between NDUFC2-silenced and control cells, with significant changes defined as ≥2.5-fold difference .

• Western Blot Analysis of Key Inflammatory Markers: Focus on:

  • NF-κB pathway components (particularly p65)

  • Phosphorylated MAP kinases (p38 MAPK and JNK1)

  • Antioxidant enzymes (SOD2)

• Subcellular Localization Studies: Track nuclear translocation of NF-κB using cell fractionation followed by western blotting or immunofluorescence microscopy.

• Intervention Studies: Test whether antioxidants or specific anti-inflammatory compounds can rescue phenotypes associated with NDUFC2 deficiency.

The search results indicate that NDUFC2 inhibition is associated with increased inflammation both in vitro and in vivo, suggesting a mechanistic link between mitochondrial dysfunction and inflammatory activation .

What are common pitfalls when working with NDUFC2 antibodies in mitochondrial research?

Researchers should be aware of these potential issues when working with NDUFC2 antibodies:

• Epitope Masking: NDUFC2 exists in a multi-protein complex with numerous protein-protein interactions that may obscure antibody binding sites. The search results show NDUFC2 interacts with at least 12 other subunits, potentially limiting epitope accessibility .

• Extraction Method Sensitivity: Depending on the detergents used, complex I may dissociate into subcomplexes, altering NDUFC2 detection patterns. Consider using milder detergents (digitonin) for structural studies versus stronger detergents (Triton X-100) for maximum extraction.

• Expression Level Variations: NDUFC2 expression levels may vary significantly between tissues and cell types, requiring optimization of antibody dilutions for each experimental system.

• Cross-Reactivity Concerns: Antibodies may cross-react with other small mitochondrial membrane proteins. Always validate specificity using appropriate controls including NDUFC2-knockout or silenced samples.

• Sample Preparation Effects: Mitochondrial proteins are sensitive to freeze-thaw cycles and oxidation. Maintain consistent sample handling procedures to ensure reproducible results.

How can researchers optimize detection of NDUFC2 in various experimental contexts?

For optimal detection of NDUFC2 across experimental systems:

• Extraction Buffer Optimization:

  • For whole-cell extracts: Use buffers containing 1-2% Triton X-100 with protease inhibitors

  • For maintaining complex integrity: Use 1% digitonin or 0.5-1% DDM (n-dodecyl β-D-maltoside)

• Gel System Selection:

  • For size determination: Standard SDS-PAGE (12-15% gels recommended for this small protein)

  • For complex integrity: Blue Native PAGE (gradient gels of 3-12%)

• Transfer Conditions:

  • Optimize transfer time and voltage for this small protein (typically higher voltage for shorter time)

  • Consider using PVDF membranes with 0.2μm pore size rather than 0.45μm for better retention

• Signal Enhancement:

  • Consider using signal amplification systems for low abundance detection

  • Optimize blocking conditions to reduce background while preserving specific signal

• Antibody Validation:

  • Test multiple commercial antibodies targeting different epitopes

  • Validate with positive controls (tissues with high mitochondrial content) and negative controls (NDUFC2-silenced samples)

How can NDUFC2 antibodies be employed in investigating the role of this protein in disease models beyond mitochondrial disorders?

NDUFC2 antibodies can be valuable tools for investigating this protein's role in diverse disease contexts:

• Neurodegenerative Disorders: Beyond Leigh syndrome (directly linked to NDUFC2 mutations), investigate potential NDUFC2 alterations in Parkinson's, Alzheimer's, and ALS models using immunohistochemistry and biochemical approaches.

• Stroke Research: The search results indicate associations between NDUFC2 variants and stroke susceptibility, with the T allele variant at NDUFC2/rs11237379 linked to early-onset ischemic stroke . Antibody-based approaches can help characterize NDUFC2 expression in relevant tissues from animal models and patient samples.

• Cancer Metabolism Studies: Examine NDUFC2 expression and complex I alterations in tumors using tissue microarrays and immunostaining.

• Aging Research: Investigate age-related changes in NDUFC2 levels and complex I assembly across tissues.

• Inflammatory Conditions: The search results suggest connections between NDUFC2 dysfunction and inflammatory pathway activation . NDUFC2 antibodies can help characterize this relationship in models of inflammatory diseases.

For these applications, consider using multiplexed immunofluorescence approaches to correlate NDUFC2 expression with cell type markers, indicators of mitochondrial function, and disease-specific pathological features.

What methodological approaches can be used to study post-translational modifications of NDUFC2?

To investigate post-translational modifications (PTMs) of NDUFC2:

• PTM-Specific Antibodies: Utilize antibodies targeting common PTMs (phosphorylation, acetylation, ubiquitination) in combination with NDUFC2 antibodies.

• Mass Spectrometry Approaches:

  • Immunoprecipitate NDUFC2 using validated antibodies

  • Process samples for LC-MS/MS analysis

  • Use database searches incorporating potential modifications

  • Consider enrichment strategies for specific modifications

• Site-Directed Mutagenesis: Generate constructs with mutations at predicted modification sites and assess effects on complex I assembly using antibody-based detection methods.

• Pharmacological Interventions: Use inhibitors of specific PTM-related enzymes (kinases, phosphatases, acetyltransferases, deacetylases) and monitor NDUFC2 migration pattern changes via western blotting.

• 2D-PAGE Analysis: Combine isoelectric focusing with SDS-PAGE to separate NDUFC2 species with different modifications, followed by immunoblotting.

While the search results don't specifically discuss PTMs of NDUFC2, understanding these modifications could provide insights into regulatory mechanisms affecting complex I assembly and function in different physiological and pathological contexts.