NDUFAF3 Antibody

What is NDUFAF3 and why is it significant in mitochondrial research?

NDUFAF3 (formerly known as C3ORF60) is an essential factor for the assembly of mitochondrial NADH:ubiquinone oxidoreductase complex (complex I) . Research has demonstrated that NDUFAF3 is a genuine mitochondrial complex I assembly protein that interacts with complex I subunits and tightly associates with NDUFAF4 (C6ORF66), another protein implicated in complex I deficiency . The significance of NDUFAF3 was highlighted when mutations in the gene were identified in patients with complex I deficiency, establishing it as a critical factor in mitochondrial function and disease pathogenesis . Gene conservation analysis has further linked NDUFAF3 to bacterial membrane insertion gene cluster SecF/SecD/YajC and to C8ORF38, which is also implicated in complex I deficiency .

What applications are NDUFAF3 antibodies most commonly used for?

NDUFAF3 antibodies are utilized across multiple molecular and cellular techniques:

These applications enable researchers to study NDUFAF3 expression patterns, subcellular localization, protein-protein interactions, and alterations in disease states .

How specific are commercial NDUFAF3 antibodies for detecting human versus other species' proteins?

Commercial NDUFAF3 antibodies vary in their species reactivity profiles. Some antibodies are specifically developed for human NDUFAF3 detection, while others demonstrate cross-reactivity with mouse or zebrafish homologs . For example, antibody ABIN7160882 is reported to react with human samples, while ABIN654942 recognizes both human and mouse NDUFAF3 . Species-specific validation is crucial when selecting antibodies for comparative studies, particularly when investigating evolutionary conservation of complex I assembly mechanisms. Sequence alignment analysis between species can help predict potential cross-reactivity, but empirical validation is necessary for confirming specificity in experimental systems.

What controls should be included when using NDUFAF3 antibodies in immunoblotting experiments?

When designing immunoblotting experiments with NDUFAF3 antibodies, several controls are essential for result validation:

• Positive controls: Lysates from cells known to express NDUFAF3 (e.g., RT-4, U251-MG cell lines)

• Negative controls:

  • Lysates from cells where NDUFAF3 has been knocked down via siRNA (siRNAs targeting NDUFAF3: 5′-AUGUAAGUGAAGUCCCUCC dTdT-3′ and 5′-AGGAAGUUGAAGGUGGCAC dTdT-3′ have been used successfully)

  • Samples from tissues with minimal NDUFAF3 expression

• Loading controls: Antibodies against housekeeping proteins (e.g., β-actin, GAPDH) or mitochondrial markers (e.g., SDHA, COX5A) to normalize expression levels

• Antibody specificity controls: Pre-absorption with the immunizing peptide (for antibodies raised against peptides APRRGHRLSPADDELY or RQRGIAVEVQDTPNAC)

The expected molecular weight for NDUFAF3 is approximately 20 kDa , and bands should be verified at this size to confirm specific detection.

How can researchers optimize immunohistochemistry protocols for NDUFAF3 detection in tissue samples?

Optimizing immunohistochemistry for NDUFAF3 requires attention to several methodological aspects:

• Fixation and antigen retrieval: Paraffin-embedded tissues typically require heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Antibody dilution: Begin with manufacturer-recommended dilutions (e.g., 1/50 for ab122270 in IHC-P applications)

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature

• Detection systems: HRP-conjugated secondary antibodies with DAB visualization provide good contrast for mitochondrial proteins

• Counterstaining: Hematoxylin counterstaining helps visualize tissue architecture while differentiating NDUFAF3-specific signals

Validation across multiple tissue types (e.g., testis, prostate, kidney) can provide confidence in protocol optimization, as NDUFAF3 expression patterns may vary between tissues .

How can researchers employ co-immunoprecipitation to investigate NDUFAF3 interactions with other complex I assembly factors?

NDUFAF3 functions in conjunction with other assembly factors, particularly NDUFAF4, making co-immunoprecipitation (Co-IP) a valuable technique for studying these interactions:

• Lysate preparation: Mitochondrial enrichment is recommended before solubilization with mild detergents (e.g., n-dodecyl-β-D-maltoside or digitonin) to preserve native protein interactions

• Antibody selection: Use purified antibodies against NDUFAF3 or epitope-tagged versions (e.g., NDUFAF3-TAP or NDUFAF3-GFP constructs)

• Crosslinking considerations: Reversible crosslinkers can stabilize transient interactions

• Washing stringency: Balance between removing non-specific interactions and preserving genuine but weak interactions

• Detection methods: Western blotting with antibodies against potential interacting partners (e.g., NDUFAF4, NDUFS3, ND1, NDUFS2)

Tandem affinity purification (TAP) combined with mass spectrometry has been successfully employed to identify novel NDUFAF3 interaction partners, providing insights into complex I assembly intermediate composition .

What approaches can be used to investigate the dynamics of NDUFAF3 localization during mitochondrial stress?

The dynamic localization of NDUFAF3 under various stress conditions can be investigated through several complementary approaches:

• Live-cell imaging: NDUFAF3-GFP fusion proteins expressed in HEK293 cells can be used alongside mitochondrial markers like Mitotracker Red for real-time visualization of localization changes

• Subcellular fractionation: Differential centrifugation to separate mitochondrial fractions from cytosolic components, followed by Western blotting

• Stress induction protocols:

  • Oxidative stress (hydrogen peroxide, paraquat)

  • Mitochondrial membrane potential disruptors (CCCP, antimycin A)

  • Complex I inhibitors (rotenone)

• Quantitative analysis: Image analysis software to measure co-localization coefficients with mitochondrial markers

• Temporal resolution: Time-course experiments to capture transient localization changes

Comparing results across multiple stress conditions can provide insights into how NDUFAF3 trafficking responds to specific mitochondrial perturbations, potentially revealing regulatory mechanisms of complex I assembly under stress .

How should researchers interpret conflicting results when studying NDUFAF3 and NDUFAF4 interactions?

Conflicting results in NDUFAF3-NDUFAF4 interaction studies may arise from several factors:

• Experimental system differences:

  • Cell lines vs. patient-derived fibroblasts

  • Endogenous vs. overexpressed proteins

  • Species-specific variations

• Technical variables:

  • Antibody specificity and epitope accessibility

  • Buffer conditions affecting protein conformation

  • Detergent selection impacting membrane protein solubilization

• Resolution approaches:

  • Employ multiple, complementary techniques (e.g., Co-IP, proximity ligation assay, FRET)

  • Verify with reciprocal experiments (pull-down with anti-NDUFAF3 then detect NDUFAF4 and vice versa)

  • Utilize siRNA knockdown of either factor to observe effects on the other (siRNAs targeting NDUFAF4: 5′-UGGAUAGAGACUAAUCUGC dTdT-3′ and 5′-AUCUUUGGAAUCAACAUAC dTdT-3′)

  • Structure-function analysis with truncated or mutated variants

Studies have demonstrated that NDUFAF3 tightly interacts with NDUFAF4 during complex I assembly , but the precise stoichiometry and temporal dynamics of this interaction may vary depending on cellular context and assembly stage.

What strategies can resolve non-specific binding issues when using NDUFAF3 antibodies in Western blotting?

Non-specific binding is a common challenge when working with mitochondrial protein antibodies. For NDUFAF3 Western blots, consider these troubleshooting approaches:

• Blocking optimization:

  • Test different blocking agents (5% BSA, 5% non-fat milk, commercial blockers)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Tween-20 to blocking and antibody incubation solutions

• Antibody dilution and incubation:

  • Test serial dilutions (e.g., 1/100, 1/250, 1/500, 1/1000)

  • Switch from room temperature to 4°C overnight incubation

  • Add reducing agents (2-5 mM DTT) to minimize antibody aggregation

• Stringency adjustments:

  • Increase salt concentration in wash buffers (from 150 mM to 300 mM NaCl)

  • Add 0.1% SDS to wash buffers for more stringent conditions

  • Increase number and duration of washing steps

• Sample preparation refinements:

  • Enrich for mitochondrial fractions to increase target:background ratio

  • Add protease inhibitors to prevent degradation products

  • Optimize protein loading (10-40 μg recommended)

The expected NDUFAF3 band size is 20 kDa . Validation across multiple sample types (e.g., RT-4, U251-MG lysates, human plasma, liver, and tonsil lysates) can help identify consistent specific signals versus non-specific artifacts .

How can researchers verify antibody specificity for NDUFAF3 versus homologous proteins?

Verifying NDUFAF3 antibody specificity against homologous proteins requires systematic validation approaches:

• Sequence-based analysis:

  • Perform BLAST analysis of the immunizing peptide against the proteome

  • Identify proteins with significant sequence similarity to NDUFAF3

  • Predict potential cross-reactivity based on epitope conservation

• Experimental validation:

  • Overexpression systems: Test antibody against cells expressing NDUFAF3-GFP fusion proteins

  • Knockdown validation: Confirm signal reduction in NDUFAF3 siRNA-treated samples

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Cross-reactivity assessment:

  • Test against recombinant homologous proteins

  • Analyze tissues with differential expression of NDUFAF3 and homologs

  • Compare staining patterns with multiple antibodies targeting different NDUFAF3 epitopes

• Mass spectrometry confirmation:

  • Perform immunoprecipitation followed by liquid chromatography and FT-MS analysis to identify all proteins captured by the antibody

This multi-layered approach ensures that signals detected with NDUFAF3 antibodies genuinely represent the target protein rather than homologous proteins or non-specific binding.

How can NDUFAF3 antibodies be used to investigate the sequential assembly of complex I modules?

NDUFAF3 antibodies are valuable tools for studying the modular assembly process of mitochondrial complex I:

• Blue Native PAGE analysis:

  • Use one-dimensional BN-PAGE (5%-15% gradient) followed by in-gel complex I activity assays to visualize assembly intermediates

  • Combine with second-dimension SDS-PAGE and immunoblotting with NDUFAF3 antibodies to identify specific subcomplexes containing this assembly factor

• Pulse-chase experiments:

  • Track the incorporation of newly synthesized subunits into intermediates

  • Immunoprecipitate NDUFAF3-containing complexes at different time points

  • Analyze the composition of these complexes to establish assembly sequence

• Genetic perturbation approaches:

  • siRNA knockdown of NDUFAF3 (using established sequences: 5′-AUGUAAGUGAAGUCCCUCC dTdT-3′ and 5′-AGGAAGUUGAAGGUGGCAC dTdT-3′)

  • Monitor accumulation or depletion of specific intermediates

  • Combine with co-immunoprecipitation to identify altered interactions

• Patient-derived cell studies:

  • Compare complex I assembly in cells from patients with NDUFAF3 mutations versus controls

  • Perform complementation with wild-type NDUFAF3 to rescue assembly defects

These approaches have revealed that NDUFAF3 acts early in the assembly process, cooperating with NDUFAF4 to facilitate the incorporation of specific subunits into growing complex I subcomplexes .

What considerations should researchers take into account when designing experiments to investigate NDUFAF3 mutations in patient samples?

When investigating NDUFAF3 mutations in patient samples, several important considerations must be addressed:

• Genetic analysis approaches:

  • Comprehensive sequencing of all exons and splice sites, as different mutations have been identified in patients

  • Homozygosity mapping may help identify candidate mutations in consanguineous families

  • Consider analyzing all three alternative spliced exons of exon 1 for complete coverage

• Functional validation strategies:

  • Complementation studies using baculovirus expressing wild-type NDUFAF3-GFP in patient fibroblasts

  • Measure complex I activity before and after complementation

  • Analyze assembly intermediate accumulation patterns

• Control selection:

  • Age-matched controls are essential

  • Consider using patient cells complemented with wild-type NDUFAF3 as internal controls

  • Include patients with other complex I deficiencies for comparison

• Technical adaptations:

  • Limited patient material may require protocol miniaturization

  • Consider immortalization options for fibroblasts to create renewable resources

  • Parallel analysis of multiple biochemical parameters from single samples

• Ethical considerations:

  • Ensure proper institutional approval for experiments involving patient materials

  • Maintain patient privacy and data confidentiality

  • Consider returning research results if clinically actionable findings emerge

These considerations help ensure robust, reproducible, and ethically sound research on pathogenic NDUFAF3 mutations and their impact on complex I assembly and function.