NDUFB3 Antibody

What is NDUFB3 and why is it important in mitochondrial research?

NDUFB3 (NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3) is an accessory subunit of the mitochondrial respiratory chain Complex I. While not directly involved in catalysis, it plays a crucial role in maintaining the structural integrity and functionality of Complex I. This complex is essential for electron transfer from NADH to ubiquinone in the mitochondrial respiratory chain, making it vital for cellular energy production .

Complex I dysfunction is implicated in numerous mitochondrial disorders, making NDUFB3 antibodies valuable tools for investigating mitochondrial pathologies. Mutations in the NDUFB3 gene have been associated with mitochondrial complex I deficiency (particularly Nuclear Type 25) and other mitochondrial disorders .

What types of NDUFB3 antibodies are available for research applications?

Several types of NDUFB3 antibodies are available for research applications:

Each antibody type offers specific advantages depending on your experimental design and research goals .

How should I optimize NDUFB3 antibody dilutions for Western blot applications?

Optimal dilution of NDUFB3 antibodies for Western blot varies based on the specific antibody and experimental conditions. Based on validated protocols:

• Initial testing range: Start with manufacturer-recommended dilutions (typically 1:500-1:2000 for polyclonal and 1:1000 for monoclonal antibodies)

• Sample loading: Use 10-40 μg of total protein per lane (20 μg is often optimal)

• Gel selection: 12% SDS-PAGE is ideal for separating the ~11 kDa NDUFB3 protein

• Transfer conditions: Standard transfer protocols for small proteins are suitable

• Blocking: 5% non-fat dry milk in TBST is effective for most NDUFB3 antibodies

• Exposure time: Begin with 30 seconds and adjust as needed

For reference, antibody M12461 showed clear bands at 1:8000 dilution with 20 μg of human heart, mouse brain, and mouse heart lysates, while antibody ab202585 worked well at 1:1000 dilution with 10 μg of human fetal tissue lysates .

What protocols should I follow for immunohistochemical detection of NDUFB3?

For optimal immunohistochemical detection of NDUFB3:

• Tissue preparation: Use either frozen sections or formalin-fixed, paraffin-embedded tissues

• Section thickness: 10 μm sections are recommended for better visualization

• Antigen retrieval:

  • Primary option: TE buffer at pH 9.0

  • Alternative: Citrate buffer at pH 6.0

• Antibody dilution: 1:50-1:500 for most polyclonal antibodies

• Co-staining markers: For mitochondrial studies, consider co-staining with:

  • Porin (mitochondrial mass marker)

  • COX-I (Complex IV marker)

  • Laminin (tissue structure marker)

• Detection systems: Standard HRP/DAB systems or fluorescent secondary antibodies

When using quadruple immunofluorescent assays, protocols validated in mitochondrial research include antibodies against NDUFB8, porin, COX-I, and laminin for comprehensive mitochondrial assessment .

How can NDUFB3 antibodies be used to assess Complex I assembly defects in mitochondrial disease?

Assessment of Complex I assembly defects using NDUFB3 antibodies involves multi-faceted approaches:

• Immunohistochemical quadruple assay:

  • Combine anti-NDUFB3 with antibodies against porin (mitochondrial mass), COX-I (Complex IV), and laminin (fiber boundary)

  • This allows quantification of NDUFB3 levels relative to mitochondrial mass in individual cells

• Blue Native PAGE (BN-PAGE):

  • Used to evaluate steady-state levels of fully assembled Complex I (980 kDa)

  • NDUFB3 antibodies can detect specific assembly intermediates

  • Compare against Complex II (detected with anti-SDHA) as an internal control

• Data interpretation:

  • Normal NDUFB3 immunoreactivity with decreased Complex I activity suggests catalytic defects

  • Decreased NDUFB3 immunoreactivity indicates assembly or stability defects

Research has shown that patients with nuclear-encoded Complex I subunit mutations (including NDUFB3 p.Trp22Arg) demonstrate reduced NDUFB3 immunoreactivity despite variable residual Complex I activities (39%-93%), while some patients with mtDNA-encoded CI mutations may show normal NDUFB3 levels despite functional defects .

What are the methodological considerations when using NDUFB3 antibodies to study mitochondrial ROS production?

When investigating mitochondrial reactive oxygen species (mitoROS) using NDUFB3 antibodies:

• Experimental design:

  • Combine NDUFB3 expression analysis with mitoROS measurement techniques

  • Consider knockdown/overexpression experiments to establish causality

• Technical approaches:

  • Measure oxygen consumption rate (OCR) in conjunction with NDUFB3 expression

  • Assess ATP levels and Complex I activity in parallel

  • Quantify mitoROS using specific fluorescent probes

• Controls and validations:

  • Include mitochondrially encoded NADH:Ubiquinone oxidoreductase core subunit measurements

  • Validate findings in multiple cell lines (e.g., BCPAP and C643 thyroid cancer cells)

  • Confirm in vivo relevance using xenograft models

Research has demonstrated that NDUFB3 knockdown significantly reduces mitoROS levels, while NDUFB3 overexpression increases mitochondrial functions including oxygen consumption rate, ATP levels, Complex I activity, and mitoROS production in cancer cells .

What strategies can address non-specific binding or inconsistent results with NDUFB3 antibodies?

When encountering non-specific binding or inconsistent results:

• Antibody validation strategies:

  • Confirm specificity using knockout/knockdown controls

  • Test across multiple cell/tissue types with known NDUFB3 expression

  • Compare results across different antibody clones or sources

• Technical optimizations:

  • Increase blocking stringency (5% BSA or 5% NFDM in TBST)

  • Implement additional washing steps (5× 5-minute washes)

  • For Western blots, ensure complete protein denaturation for this membrane-associated protein

  • For IHC, optimize antigen retrieval conditions (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Sample handling considerations:

  • Ensure rapid tissue/cell processing to maintain mitochondrial integrity

  • Include protease inhibitors in lysis buffers

  • Maintain cold chain throughout sample preparation

For assay validation, positive controls should include tissues with high mitochondrial density such as heart, liver, or brain tissue, where clear NDUFB3 signals have been documented .

How can I confirm the specificity of my NDUFB3 antibody results?

To confirm NDUFB3 antibody specificity:

• Multi-antibody validation:

  • Compare results using antibodies targeting different epitopes of NDUFB3

  • Use both monoclonal and polyclonal antibodies as complementary approaches

• Alternative Complex I markers:

  • Compare with other Complex I subunit antibodies (e.g., NDUFS3)

  • Different subunits may show distinct patterns in assembly defects

• Functional correlation:

  • Correlate antibody results with Complex I enzyme activity measurements

  • Combine with BN-PAGE assessment of fully assembled Complex I

• Genetic controls:

  • When available, use samples with known NDUFB3 mutations

  • The p.Trp22Arg NDUFB3 variant has been well-characterized in patients with distinctive clinical features including short stature and specific facial characteristics

Research has shown that while NDUFB3 antibodies may fail to detect certain Complex I defects (particularly those caused by mtDNA mutations), combining with other approaches provides comprehensive assessment of Complex I status .

How do NDUFB3 antibodies contribute to phenotype-genotype correlation studies in mitochondrial diseases?

NDUFB3 antibodies have proven valuable in establishing phenotype-genotype correlations:

• Distinctive phenotypic markers:

  • The p.Trp22Arg NDUFB3 mutation causes a recognizable clinical syndrome with:

    • Short stature (<9th centile)

    • Distinctive facial features (prominent forehead, smooth philtrum, deep-set eyes)

    • Variable biochemical phenotypes

    • Often found in patients of Irish ancestry

• Biochemical correlation:

  • Patients with the p.Trp22Arg mutation show variable NDUFB3 immunoreactivity (39%-93%)

  • This correlates with differences in steady-state NDUFB3 protein levels on Western blot

  • Despite variable protein levels, clinical outcomes are often more favorable than predicted

• Diagnostic pathway optimization:

  • Recognition of characteristic facies can guide genetic testing

  • NDUFB3 antibody findings may reduce the need for muscle biopsy in some cases

Importantly, long-term prognosis for patients with NDUFB3 mutations can be good, even for those presenting with acute metabolic crisis, highlighting the value of accurate molecular diagnosis .

What role do NDUFB3 antibodies play in cancer research, particularly regarding mitochondrial function?

NDUFB3 antibodies have revealed important connections between mitochondrial function and cancer:

• Biomarker potential:

  • NDUFB3 expression levels correlate with clinical outcomes in certain cancers

  • Low NDUFB3 expression is associated with unfavorable prognosis in thyroid cancer

• Mechanistic insights:

  • NDUFB3 modulates mitochondrial reactive oxygen species (mitoROS) levels

  • NDUFB3 knockdown reduces mitoROS production

  • NDUFB3 overexpression increases:

    • Oxygen consumption rate

    • ATP production

    • Complex I activity

    • mitoROS levels

• Therapeutic implications:

  • NDUFB3 expression affects tumor growth in xenograft models

  • Targeting NDUFB3 or mitoROS pathways represents a potential therapeutic strategy

Analysis of thyroid cancer patient data revealed that patients with lower NDUFB3 expression showed predominant changes in oxidative phosphorylation pathways, highlighting the functional significance of NDUFB3 in cancer metabolism .

How can NDUFB3 antibodies be integrated into multi-omics approaches to study mitochondrial dysfunction?

Integration of NDUFB3 antibodies into multi-omics approaches:

• Proteomics integration:

  • Use NDUFB3 antibodies for immunoprecipitation followed by mass spectrometry

  • Identify novel interaction partners and post-translational modifications

  • Compare NDUFB3 interactome across different disease states

• Transcriptomics correlation:

  • Correlate NDUFB3 protein levels with gene expression profiles

  • Identify regulatory networks affected by NDUFB3 dysfunction

  • Studies have shown significant pathway changes in oxidative phosphorylation based on NDUFB3 expression levels

• Metabolomics association:

  • Link NDUFB3 levels to metabolic signatures

  • Focus on NAD+/NADH ratios and downstream metabolites

  • Investigate connections to cellular energy status

• Spatial proteomics:

  • Utilize advanced imaging with NDUFB3 antibodies to assess mitochondrial dynamics

  • Combine with mitochondrial markers to assess organelle morphology and distribution

These integrated approaches can provide comprehensive insights into how NDUFB3 dysfunction contributes to disease pathogenesis beyond isolated Complex I deficiency .

What emerging technologies are enhancing the utility of NDUFB3 antibodies in mitochondrial research?

Emerging technologies enhancing NDUFB3 antibody applications:

• Quantitative immunofluorescence techniques:

  • Quadruple immunofluorescent assays permit precise quantification of NDUFB3 relative to mitochondrial mass

  • This allows detection of subtle defects that might be missed by conventional biochemical assays

  • Statistical tools can categorize fibers as normal, intermediate, or deficient

• Super-resolution microscopy:

  • Techniques like STORM and STED provide nanoscale resolution of NDUFB3 localization

  • Enables visualization of Complex I distribution within mitochondrial cristae

• Live-cell imaging:

  • Development of cell-permeable NDUFB3 antibody fragments or nanobodies

  • Allows real-time monitoring of Complex I dynamics

• Single-cell proteomics:

  • Combines flow cytometry with NDUFB3 antibodies

  • Permits analysis of Complex I heterogeneity within tissues

  • Enables correlation with other cellular parameters

• Proximity labeling techniques:

  • APEX2 or BioID fusions with NDUFB3

  • Maps protein neighborhoods within the mitochondrial membrane

  • Identifies transient interactions during Complex I assembly