NDUFS6 Antibody

What is NDUFS6 and why is it important in mitochondrial research?

NDUFS6 (NADH dehydrogenase [ubiquinone] iron-sulfur protein 6) is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). This 14 kDa protein plays a critical role in the assembly and function of complex I, which is essential for oxidative phosphorylation. The importance of NDUFS6 in research stems from its association with severe mitochondrial disorders. Bi-allelic NDUFS6 variants have been linked to lethal infantile mitochondrial disease (LMID) and Leigh syndrome (LS), with recent findings expanding the clinical spectrum to include milder phenotypes such as axonal neuropathy with optic atrophy and intellectual disability . As a complex I component, NDUFS6 provides a valuable target for studying mitochondrial function in both normal and disease states.

How does NDUFS6 deficiency affect mitochondrial function across different tissues?

Tissue-specific differences in NDUFS6 expression and complex I assembly create variable patterns of mitochondrial dysfunction. Studies using NDUFS6-deficient mouse models have demonstrated that the heart is particularly vulnerable to NDUFS6 deficiency, showing marked reduction in complex I activity (approximately 10% of normal levels). Other tissues maintain higher residual activity due to tissue-specific mRNA splicing that allows for small amounts (1-34%) of wild-type NDUFS6 mRNA to be expressed . This differential expression explains why cardiac abnormalities often predominate in both animal models and human patients with NDUFS6 mutations. Interestingly, mitochondrial copy number may remain unaltered despite NDUFS6 deficiency, as demonstrated in leukocytes from affected patients .

What criteria should researchers consider when selecting an NDUFS6 antibody?

Selecting the appropriate NDUFS6 antibody requires careful consideration of several factors:

• Species reactivity: Confirm the antibody recognizes NDUFS6 in your species of interest. For example, commercial antibodies like DF9671 react with human, mouse, and rat NDUFS6, with predicted reactivity to other vertebrates including zebrafish and bovine samples .

• Application compatibility: Verify the antibody has been validated for your specific application (Western blot, immunofluorescence, immunohistochemistry, etc.).

• Clonality: Polyclonal antibodies may offer better sensitivity by recognizing multiple epitopes, while monoclonal antibodies provide higher specificity.

• Epitope location: Consider whether the epitope is accessible in your experimental conditions, particularly for native protein detection.

• Validation data: Review the manufacturer's validation data including Western blot images showing a single band at the expected 14 kDa size .

What controls should be included when validating an NDUFS6 antibody?

Proper validation of NDUFS6 antibodies requires multiple controls:

• Positive tissue controls: Heart tissue typically shows high NDUFS6 expression and serves as an excellent positive control.

• Genetic controls: If available, samples from NDUFS6 knockout/knockdown models provide definitive negative controls. The gene-trap NDUFS6 mouse model shows tissue-specific reduction in NDUFS6 protein levels .

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide should eliminate specific binding.

• Cross-reactivity assessment: Test the antibody against recombinant NDUFS6 and related complex I subunits to confirm specificity.

• Size verification: Confirm detection of a single band at the expected molecular weight (14 kDa) in Western blots .

What are the optimal conditions for Western blot detection of NDUFS6?

Western blot detection of NDUFS6 requires specific conditions for optimal results:

• Sample preparation: Mitochondrial enrichment significantly improves detection of this low-abundance protein. Fresh samples yield better results as NDUFS6 can degrade during storage.

• Protein separation: Use 12-15% polyacrylamide gels to properly resolve this small (14 kDa) protein .

• Transfer conditions: Transfer to PVDF membranes at lower voltage (15-20V) for longer periods (1-2 hours) often improves transfer efficiency of small proteins.

• Blocking: 5% non-fat milk in TBST is typically effective, though some antibodies may perform better with BSA-based blocking buffers.

• Antibody dilution: Commercial NDUFS6 antibodies like DF9671 typically work well at 1:1000 to 1:2000 dilutions, but optimal concentrations should be determined empirically .

• Detection system: Enhanced chemiluminescence systems provide suitable sensitivity for detecting endogenous NDUFS6 levels.

How can NDUFS6 antibodies be used to study complex I assembly defects?

NDUFS6 antibodies are valuable tools for investigating complex I assembly:

• Blue Native PAGE (BN-PAGE): This technique preserves native protein complexes and can reveal assembly intermediates. Subsequent immunoblotting with NDUFS6 antibodies can identify which subcomplexes contain this protein. Studies have shown that NDUFS6 deficiency affects the migration pattern of complex I in BN-PAGE .

• Immunoprecipitation: NDUFS6 antibodies can pull down interaction partners during complex I assembly, helping to define the protein's role in the assembly process.

• Immunofluorescence co-localization: Co-staining with other complex I subunits can reveal spatial relationships during assembly and identify mislocalization in pathological states.

• Proteomic profiling: As demonstrated in recent research, proteomic analysis of cells with NDUFS6 deficiency can identify broader changes in protein expression. In one study, loss of NDUFS6 was associated with decreased levels of three other mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4, and NDUFV1) .

How can NDUFS6 antibodies contribute to research on neuropathies and mitochondrial diseases?

NDUFS6 antibodies provide valuable insights into the pathophysiology of neuropathies and mitochondrial diseases:

• Biomarker validation: Research has investigated correlations between NDUFS6 deficiency and mitochondrial biomarkers such as GDF15, though not all biomarkers show significant changes in NDUFS6-related disorders .

• Tissue-specific pathology: Immunohistochemistry with NDUFS6 antibodies can reveal differential expression patterns in affected versus unaffected tissues in patients with mitochondrial disorders.

• Therapeutic development: Monitoring NDUFS6 protein levels can help assess the efficacy of experimental therapies aimed at rescuing complex I deficiency.

• Genotype-phenotype correlations: Combined with genetic information, NDUFS6 protein analysis can help explain why some mutations cause severe disease (LMID, Leigh syndrome) while others result in milder phenotypes like axonal neuropathy .

• Skin biopsy analysis: In patients with neuropathy, correlating NDUFS6 expression with intraepidermal nerve fiber density may provide insights into pathophysiology, as reduced density (3.8 fibers/mm compared to normal 6.1-10.9 fibers/mm) has been observed in NDUFS6-deficient patients .

What proteomic approaches can be combined with NDUFS6 antibodies for comprehensive mitochondrial studies?

Integrating NDUFS6 antibodies with proteomics enhances mitochondrial research:

• Data-independent proteomic profiling: This approach has successfully quantified over 3,500 proteins in white blood cells from patients with NDUFS6 mutations, identifying 36 significantly dysregulated proteins (1.02% of quantified proteins) .

• Immunocapture-MS: Using NDUFS6 antibodies to pull down protein complexes followed by mass spectrometry can identify novel interaction partners.

• Quantitative Western blotting: Combining immunoblotting with proteomics provides targeted validation of global proteomic findings.

• Spatial proteomics: Correlating NDUFS6 localization with proteomic changes in specific subcellular compartments can reveal organelle crosstalk in disease states.

• Gene Ontology analysis: In silico analysis of proteomics data from NDUFS6-deficient samples has identified altered mitochondrial respiratory chain complex I assembly as a key affected pathway .

What are common challenges when using NDUFS6 antibodies and how can they be addressed?

Researchers frequently encounter these issues when working with NDUFS6 antibodies:

• Low signal intensity:

  • Increase antibody concentration or incubation time

  • Perform mitochondrial enrichment before analysis

  • Use more sensitive detection systems

  • Consider tissue-specific expression levels (heart samples typically show stronger signals)

• Non-specific bands:

  • Increase blocking time or concentration

  • Titrate primary antibody to optimal concentration

  • Use more stringent washing conditions

  • Consider alternative antibodies with different epitope recognition

• Variability between experiments:

  • Standardize protein extraction methods

  • Include loading controls specific for mitochondrial proteins

  • Use consistent sample handling procedures

  • Validate results with multiple antibodies when possible

• Difficult detection in certain tissues:

  • Consider tissue-specific splicing that may affect epitope expression

  • Adapt protocols for tissues with lower NDUFS6 expression

  • Use gene-trap models as reference points for expected variability

How can researchers distinguish between normal and pathogenic alterations in NDUFS6 expression?

Distinguishing pathogenic from normal variations requires these approaches:

• Quantitative analysis: Compare NDUFS6 expression levels across multiple controls and affected samples using densitometry or fluorescence quantification.

• Functional correlation: Link NDUFS6 levels to complex I activity measurements using biochemical assays.

• Context interpretation: Consider genetic background, tissue type, and developmental stage when interpreting NDUFS6 expression patterns.

• Comparative proteomics: Look for concomitant changes in other complex I subunits, particularly NDUFA12, NDUFS4, and NDUFV1, which are known to decrease alongside NDUFS6 deficiency .

• Reference range establishment: Develop laboratory-specific reference ranges for different tissues and experimental conditions.