NDUFB6 Antibody

What is NDUFB6 and why is it significant in mitochondrial research?

NDUFB6 is a subunit of the ND5-module of mitochondrial Complex I, the largest complex in the respiratory chain. This protein has emerged as an important research target for several reasons:

• It demonstrates remarkable stability even when other Complex I components are depleted or absent

• It can form stable subcomplexes during Complex I assembly processes

• Its downregulation has been implicated in certain cancers, notably renal cell carcinoma

• It serves as a valuable marker for studying Complex I assembly dynamics and dysfunction

Research has demonstrated that NDUFB6 levels remain consistently stable even after virtually complete repression of other CI subunits like NDUFS3, while subunits belonging to the N- and Q-modules (such as NDUFA12 and NDUFS6) show prompt decreases in their steady-state levels .

What are the recommended applications and working concentrations for NDUFB6 antibodies?

Based on validated research protocols, NDUFB6 antibodies have been successfully employed in multiple experimental techniques with the following optimized parameters:

For detection in Western blot analyses, ECL Prime Western Blotting Detection Reagents have been successfully used, with GAPDH serving as an effective loading control .

How can NDUFB6 antibodies be utilized to study Complex I assembly dynamics?

NDUFB6 antibodies offer unique insights into Complex I assembly due to the protein's stability characteristics:

• Blue Native PAGE (BN-PAGE) analysis: Following NDUFS3 depletion, studies have documented the accumulation of subcomplexes containing NDUFB8 and NDUFB6, while fully assembled CI progressively decreases . This makes NDUFB6 antibodies valuable for tracking assembly intermediates.

• Assembly defect characterization: The reduction of certain CI components (e.g., NDUFS3) leads to decreased levels of N- and Q-module subunits while NDUFB6 remains stable, suggesting differential stability of CI modules .

• Monitoring assembly dynamics: By combining NDUFB6 antibodies with antibodies against other CI subunits in time-course experiments, researchers can map the sequential incorporation of components into the complex.

• Interaction studies: NDUFB6 can be studied alongside other CI components using techniques like crosslinking mass spectrometry (XL-MS) to identify specific interaction partners, as demonstrated with other CI assembly factors .

What methodologies have been validated for studying NDUFB6 in cancer research?

Several methodological approaches have been rigorously validated for investigating NDUFB6 in cancer contexts:

• RNA interference: Specific siRNA sequences targeting NDUFB6 have been validated:

  • siRNA1: 5′-TCATGTACTTGTACCTGTCTGGATT-3′

  • siRNA2: 5′-GAGCCTAAGTTTGTTCCTATATTAC-3′

• Lentiviral overexpression: Lv-NDUFB6 constructs have been successfully used to restore NDUFB6 expression in cell lines with downregulated levels .

• Functional assays: Following NDUFB6 manipulation:

  • Cell proliferation assays show increased proliferation with NDUFB6 knockdown and suppressed proliferation with overexpression in RCC cell lines (786-O and 769-P)

  • No significant changes were observed in apoptosis, invasion, or migration capabilities

• EMT marker analysis: Expression of EMT-associated genes such as vimentin, ZEB1, and occludin can be assessed by qRT-PCR following NDUFB6 manipulation, though studies suggest NDUFB6 is primarily involved in proliferation rather than EMT processes .

What are the critical controls needed when using NDUFB6 antibodies?

To ensure experimental validity when working with NDUFB6 antibodies, researchers should implement the following controls:

• Positive controls:

  • Mitochondria-rich tissues or cell lines with known NDUFB6 expression

  • Lentiviral NDUFB6 overexpression samples

  • Recombinant NDUFB6 proteins or fragments (where applicable)

• Negative controls:

  • NDUFB6 siRNA-treated samples using validated sequences

  • Secondary antibody-only controls for immunostaining techniques

  • Non-mitochondrial cellular fractions

• Specificity validation:

  • Comparative analysis with other antibodies targeting different NDUFB6 epitopes

  • Confirmation of mitochondrial localization pattern

  • Correlation with other Complex I markers

The antibody specificity can be further verified through protein arrays, as demonstrated by testing against 364 human recombinant protein fragments in the Human Protein Atlas project .

How should experimental results be interpreted when NDUFB6 shows unexpected patterns?

When faced with unexpected NDUFB6 antibody results, consider these data interpretation guidelines:

How can I design comprehensive experiments to investigate NDUFB6's role in Complex I assembly disorders?

For thorough characterization of NDUFB6 in Complex I disorders:

• Integrative analysis approach:

  • Combine BN-PAGE with subsequent immunoblotting using antibodies against multiple CI subunits, including NDUFB6, NDUFB8, and components of N- and Q-modules

  • Correlate subcomplexes accumulation patterns with functional deficits

• Time-course manipulations:

  • Implement inducible knockdown systems for CI components

  • Track the temporal dynamics of NDUFB6-containing subcomplexes during assembly disruption

  • Analyze progressive changes in respiratory function

• Rescue experiments:

  • Test whether NDUFB6 overexpression can compensate for deficiencies in other CI components

  • Examine if other subunits can restore function in NDUFB6-deficient systems

• Structural interaction mapping:

  • Apply techniques like crosslinking mass spectrometry (XL-MS) to identify direct interaction partners of NDUFB6

  • Use yeast two-hybrid systems to confirm specific protein-protein interactions

What are methodological considerations when studying NDUFB6 in cancer progression models?

When investigating NDUFB6 in cancer contexts, consider these methodological approaches:

• Genomic analysis integration:

  • Correlate 9p24.1-p13.3 loss (where NDUFB6 is located) with clinical outcomes

  • Analyze TCGA datasets for associations between NDUFB6 copy number, expression, and patient survival

• Expression manipulation strategy:

  • Use validated siRNAs for transient knockdown experiments

  • Implement lentiviral systems for stable overexpression

  • Consider inducible systems for temporal control

• Comprehensive phenotypic assessment:

  • Primary focus: Proliferation assays (demonstrated significant effects)

  • Secondary analyses: Apoptosis, invasion, migration (no significant effects observed)

  • EMT marker analysis: vimentin, ZEB1, occludin expression by qRT-PCR

• Mitochondrial function correlation:

  • Assess respiratory capacity in relation to NDUFB6 levels

  • Measure ROS production and mitochondrial membrane potential

  • Evaluate metabolic rewiring using seahorse analysis or metabolomics

How do I reconcile contradictory findings about NDUFB6 stability in different experimental contexts?

When confronting seemingly conflicting data about NDUFB6:

• Methodological differences evaluation:

  • Cell type specificity: Different cellular backgrounds may yield varying results

  • Protein detection methods: Different antibodies may recognize distinct epitopes or conformations

  • Experimental timeframes: Acute vs. chronic manipulations may produce different outcomes

• Assembly context consideration:

  • Research clearly shows that NDUFB6 levels remain stable after virtually complete NDUFS3 repression, while other CI subunits decrease significantly

  • This suggests NDUFB6 may be incorporated into or maintained in subcomplexes independently of certain other subunits

• Validation strategy:

  • Use multiple antibodies targeting different NDUFB6 epitopes

  • Employ complementary techniques (immunoblotting, immunofluorescence, mass spectrometry)

  • Consider post-translational modifications that might affect epitope recognition

What remains unknown about NDUFB6 function that requires further investigation?

Despite significant advances, several knowledge gaps remain in NDUFB6 research:

• Precise assembly role:

  • The exact step at which NDUFB6 is incorporated into Complex I

  • Whether NDUFB6 serves as an assembly factor or solely a structural component

  • Potential chaperone interactions during the assembly process

• Cancer biology mechanisms:

  • How NDUFB6 downregulation mechanistically leads to increased cell proliferation

  • Whether this effect is mediated through altered mitochondrial function or independent pathways

  • Potential therapeutic implications of restoring NDUFB6 expression in cancers with 9p loss

• Tissue-specific functions:

  • Differential expression and significance across tissue types

  • Potential tissue-specific interaction partners

  • Varied consequences of NDUFB6 deficiency in different organs

• Post-translational regulation:

  • Whether NDUFB6 undergoes regulatory modifications

  • Protein turnover and quality control mechanisms

  • Potential compensatory mechanisms when NDUFB6 is deficient

What emerging techniques could enhance NDUFB6 research beyond current methodologies?

Several cutting-edge approaches could advance NDUFB6 research:

• Cryo-EM structural studies:

  • High-resolution structural analysis of NDUFB6 within intact Complex I

  • Visualization of NDUFB6-containing subcomplexes in assembly disorders

  • Mapping of interaction interfaces with neighboring subunits

• CRISPR-based approaches:

  • Generation of NDUFB6 knockout cell lines for definitive functional studies

  • Creation of endogenously tagged NDUFB6 for live-cell imaging

  • Base editing to introduce patient-specific mutations

• Single-cell analyses:

  • Single-cell proteomics to detect NDUFB6 heterogeneity in tissues

  • Correlation with mitochondrial function at individual cell level

  • Spatial transcriptomics to map NDUFB6 expression patterns in tissue context

• Therapeutic explorations:

  • Testing whether NDUFB6 restoration can overcome certain Complex I deficiencies

  • Investigating small molecules that might stabilize NDUFB6-containing subcomplexes

  • Exploring gene therapy approaches for NDUFB6-deficient conditions

How can NDUFB6 antibodies be integrated into mitochondrial disease diagnostic workflows?

NDUFB6 antibodies offer diagnostic potential in several contexts:

• Biomarker development:

  • Distinctive patterns of NDUFB6-containing subcomplexes may serve as fingerprints for specific Complex I assembly defects

  • Integration into diagnostic algorithms alongside established mitochondrial markers

• Tissue-based diagnostics:

  • Immunohistochemical evaluation of NDUFB6 in muscle biopsies from patients with suspected mitochondrial disorders

  • Correlation with clinical phenotypes and genetic findings

• Prognostic applications:

  • Assessment of NDUFB6 expression levels in cancer samples for potential prognostic information

  • Integration with genomic data (9p24.1-p13.3 loss) for comprehensive evaluation

• Therapeutic monitoring:

  • Using NDUFB6 antibodies to track restoration of proper Complex I assembly in response to experimental therapies

  • Development of companion diagnostics for mitochondrial-targeted interventions