NDUFA3 Antibody

What is NDUFA3 and what role does it play in mitochondrial function?

NDUFA3 (NADH Dehydrogenase Ubiquinone 1 Alpha Subcomplex, 3, 9kDa) functions as an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase, commonly known as Complex I . Unlike catalytic subunits, NDUFA3 is believed not to be directly involved in the enzymatic reaction but plays a crucial structural role in the assembly and stability of Complex I . The protein facilitates the transfer of electrons from NADH to the respiratory chain, with ubiquinone serving as the immediate electron acceptor . NDUFA3's importance is underscored by studies showing that its knockout in cultured human cells results in reduced expression of Complex I and abnormal migration patterns during analysis, suggesting essential roles in complex assembly and integrity .

NDUFA3 has a calculated molecular weight of 9 kDa (84 amino acids), though the human protein purified from mitochondria typically migrates at approximately 9-10 kDa on SDS-PAGE . When performing Western blot analysis, researchers should optimize protocols to detect this small protein effectively, as typical settings for larger proteins may not be optimal.

Regarding cellular localization, NDUFA3 is predominantly found in mitochondria as expected for a component of the respiratory chain. Immunofluorescence studies show a distinctive mitochondrial network pattern that colocalizes with other mitochondrial markers . When performing subcellular fractionation, NDUFA3 should appear enriched in mitochondrial fractions and absent from cytosolic preparations when protocols are properly executed.

What are the optimal sample preparation techniques for detecting NDUFA3 in Western blot experiments?

For effective NDUFA3 detection in Western blot experiments, consider the following protocol optimization strategies:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors for cell lysis

  • Maintain cold temperatures throughout extraction to prevent protein degradation

  • Sonicate briefly to ensure complete membrane disruption and release of mitochondrial proteins

• Gel Electrophoresis Parameters:

  • Use 12-15% polyacrylamide gels to properly resolve the small 9 kDa protein

  • Load 20-30 μg of total protein per lane for typical cell lysates

  • Include positive controls such as HeLa cell lysate, which has been validated for NDUFA3 detection

• Transfer Conditions:

  • Optimize for small proteins: use PVDF membranes with 0.2 μm pore size rather than 0.45 μm

  • Consider semi-dry transfer systems with 20% methanol in transfer buffer

  • Transfer at lower voltage (10-15V) for longer periods (60-90 mins) to prevent small proteins from passing through the membrane

Western blot antibody incubation is typically performed at dilutions of 1:500-1:1000, though this may vary between suppliers .

What epitopes of NDUFA3 are targeted by different antibodies, and how might this affect experimental outcomes?

Different commercial antibodies target various regions of the NDUFA3 protein, which can significantly impact experimental results:

For example, one specific NDUFA3 antibody targets the internal sequence CYPVPVRDDGNMPDV , while others target fusion proteins containing amino acids 2-84 . When comparing results between studies using different antibodies, researchers should consider these epitope differences as potential sources of variability.

For applications requiring detection of specific NDUFA3 variants or in tissues where post-translational modifications may be relevant, selecting antibodies targeting appropriate epitopes becomes crucial for experimental success.

What are the recommended fixation and antigen retrieval methods for NDUFA3 immunohistochemistry?

Optimal protocols for NDUFA3 immunohistochemistry include:

• Fixation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used

  • For cell cultures, 4% paraformaldehyde fixation for 15-20 minutes at room temperature

• Antigen Retrieval:

  • Heat-induced epitope retrieval using TE buffer at pH 9.0 is recommended as the primary method

  • Alternative method: citrate buffer at pH 6.0 if TE buffer produces suboptimal results

  • Typically heat at 95-100°C for 15-20 minutes followed by cooling to room temperature

• Antibody Dilution and Incubation:

  • IHC applications typically use dilutions ranging from 1:50 to 1:500

  • Overnight incubation at 4°C generally yields optimal signal-to-noise ratio

  • Use appropriate blocking (5% normal serum corresponding to secondary antibody species) to reduce background

These protocols have been validated for human tissues including liver, which shows reliable NDUFA3 immunoreactivity and can serve as a positive control tissue .

How can NDUFA3 antibodies be used to investigate mitochondrial complex I deficiencies in disease models?

NDUFA3 antibodies provide valuable tools for investigating complex I deficiencies through several methodological approaches:

• Diagnostic Applications:

  • Western blotting to assess NDUFA3 protein levels in patient-derived samples

  • Immunohistochemistry to visualize altered distribution patterns in affected tissues

  • Blue Native PAGE combined with NDUFA3 immunoblotting to assess complex I assembly defects

• Functional Assessment:

  • Co-immunoprecipitation experiments to identify altered protein-protein interactions

  • Tracking expression changes in response to oxidative stress or metabolic challenges

  • Correlation of NDUFA3 levels with complex I activity measurements and clinical phenotypes

• Research Applications in Disease Models:

  • Recent studies have utilized NDUFA3 as a marker in diabetes-associated intervertebral disc degeneration

  • NDUFA3 overexpression has demonstrated protective effects against high glucose-induced cellular injuries through suppression of apoptosis, ROS elimination, and improved mitochondrial function

  • HDAC/H3K27ac mechanisms have been implicated in regulating NDUFA3 transcription, suggesting epigenetic therapeutic targets

When using NDUFA3 antibodies in disease research, complementary assays measuring mitochondrial membrane potential, oxygen consumption rate, and ROS production provide comprehensive assessment of mitochondrial dysfunction .

What validation strategies should be employed when using NDUFA3 antibodies in novel experimental systems?

When implementing NDUFA3 antibodies in new experimental systems, rigorous validation is essential:

• Antibody Specificity Controls:

  • Positive controls: Human brain tissue and HeLa cells have validated NDUFA3 expression

  • Negative controls: Primary antibody omission and isotype controls

  • Knockdown/knockout validation: siRNA or CRISPR-mediated depletion of NDUFA3 should eliminate or significantly reduce signal

  • Peptide competition assay: Pre-incubation with immunizing peptide should abolish specific binding

• Cross-Reactivity Assessment:

  • Check sequence homology between species if working with non-human models

  • NDUFA3 shows high homology across mammals: 100% in cow, dog, horse, pig, and human; 86% in mouse; 93% in rat

  • Perform Western blot with tissue from multiple species to confirm expected band patterns

• Methodological Validation:

  • Optimize antibody concentration through titration experiments

  • Compare results from multiple antibodies targeting different epitopes

  • Validate subcellular localization through co-staining with established mitochondrial markers

  • Confirm expression patterns align with known tissue distribution of NDUFA3

Documentation of these validation steps is critical for publication and reproducibility of research findings involving NDUFA3.

How can researchers optimize co-immunoprecipitation protocols for studying NDUFA3 interactions with other complex I components?

Optimizing co-immunoprecipitation (Co-IP) for NDUFA3 requires specialized approaches due to its mitochondrial localization and small size:

• Lysis Buffer Considerations:

  • Use gentle non-ionic detergents (0.5-1% digitonin or 0.5% DDM) that preserve mitochondrial protein complexes

  • Include ATP (2 mM) and GTP (1 mM) to maintain complex integrity

  • Add protease inhibitors and phosphatase inhibitors to prevent degradation

• Pre-Clearing and Antibody Selection:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • For NDUFA3 pull-down, select antibodies validated for immunoprecipitation applications

  • Consider using tagged constructs (FLAG, HA) for pull-down if direct IP is inefficient

• Critical Protocol Adaptations:

  • Extend incubation times (overnight at 4°C) to enhance NDUFA3 complex capture

  • Use crosslinking agents such as DSP (dithiobis(succinimidyl propionate)) to stabilize transient interactions

  • Employ low-salt wash buffers to preserve weak interactions

  • Elute under native conditions if downstream applications require functional complexes

• Validation of Interactions:

  • Confirm expected interactions with known complex I components as positive controls

  • Verify results with reciprocal Co-IP experiments

  • Include IgG control and input samples in all Western blot analyses

  • Consider mass spectrometry analysis of co-precipitated proteins for unbiased interaction mapping

These optimized Co-IP methods have been successfully applied to study interactions between NDUFA3 and other mitochondrial proteins in both healthy and disease contexts.

What are common issues when working with NDUFA3 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with NDUFA3 antibodies:

• Low Signal Intensity in Western Blots:

  • Problem: NDUFA3 is a small protein (9 kDa) that may transfer inefficiently or diffuse from membranes

  • Solution: Use 0.2 μm PVDF membranes, reduce methanol in transfer buffer to 10%, and consider adding SDS (0.01-0.02%) to transfer buffer for small proteins

• Non-specific Bands:

  • Problem: Additional bands appearing at unexpected molecular weights

  • Solution: Increase blocking stringency (5% BSA instead of milk), optimize antibody dilution (typically 1:500-1:1000), and include appropriate negative controls

• Variable Results Between Tissue Types:

  • Problem: Inconsistent staining patterns across different tissues

  • Solution: Optimize antigen retrieval conditions for each tissue type; for NDUFA3, comparing TE buffer (pH 9.0) versus citrate buffer (pH 6.0) is recommended

• Poor Immunohistochemical Staining:

  • Problem: Weak or diffuse staining in FFPE tissues

  • Solution: Ensure proper fixation (not over-fixed), optimize antigen retrieval time, and consider signal amplification systems; for NDUFA3, dilutions between 1:50-1:100 are recommended for IHC applications

• Antibody Cross-Reactivity:

  • Problem: Non-specific binding to proteins with similar epitopes

  • Solution: Select antibodies with validated specificity for your species of interest; NDUFA3 antibodies have varied cross-reactivity patterns with human, mouse, and rat samples

Maintaining proper storage conditions (aliquot and store at -20°C, avoid repeated freeze/thaw cycles) also significantly improves antibody performance over time .

What quality control measures should be implemented when working with NDUFA3 antibodies in mitochondrial research?

Implementing rigorous quality control is essential when studying NDUFA3 in mitochondrial research:

• Antibody Validation:

  • Confirm antibody specificity using positive controls (human brain tissue, HeLa cells)

  • Include appropriate negative controls (primary antibody omission, non-expressing tissues)

  • Perform knockdown/knockout validation when possible to confirm specificity

• Sample Preparation Quality Control:

  • Assess mitochondrial enrichment using established markers (VDAC, COX IV)

  • Monitor protein degradation through evaluation of multiple mitochondrial proteins

  • Implement consistent sample handling procedures to minimize variability

• Experimental Controls for Specific Applications:

  Application	Essential Controls
  Western Blot	Positive control (HeLa lysate), loading control (GAPDH, β-actin), molecular weight marker
  IHC	Positive control tissue (liver), negative control (primary antibody omitted), isotype control
  ELISA	Standard curve with recombinant protein, blank wells, dilution linearity assessment
  IF	Co-localization with mitochondrial markers, Z-stack acquisition to confirm genuine signals

• Documentation and Reproducibility:

  • Record complete antibody information including catalog number, lot number, and dilution

  • Document all experimental conditions in detail (e.g., exposure time for Western blots, microscope settings for imaging)

  • Perform key experiments with multiple antibody lots or sources when possible

  • Implement blinding procedures for image analysis to reduce bias

These quality control measures ensure reliable and reproducible results when studying NDUFA3 in the context of mitochondrial biology and disease.

How can researchers distinguish between specific and non-specific signals when using NDUFA3 antibodies in complex tissue samples?

Distinguishing genuine NDUFA3 signals from artifacts in complex tissue samples requires systematic approaches:

• Implement Multiple Control Approaches:

  • Use known positive control tissues (human liver has been validated)

  • Compare adjacent tissue sections with primary antibody omission

  • Include isotype controls matching the primary antibody

  • When possible, include tissues from knockout models as gold-standard negative controls

• Utilize Technical Validations:

  • Perform peptide competition assays where primary antibody is pre-incubated with immunizing peptide

  • Compare staining patterns using multiple antibodies targeting different NDUFA3 epitopes

  • Correlate IHC findings with mRNA expression data from the same tissue type

  • Conduct Western blot analysis on the same tissue to confirm appropriate molecular weight

• Apply Advanced Imaging Techniques:

  • Employ co-localization studies with established mitochondrial markers (e.g., TOMM20, ATP5A)

  • Use super-resolution microscopy to confirm mitochondrial morphology

  • Implement spectral unmixing in cases of tissue autofluorescence

  • Conduct Z-stack imaging to distinguish genuine signals from artifactual overlay

• Signal Interpretation Guidelines:

  • Genuine NDUFA3 signals should follow mitochondrial distribution patterns

  • Signal intensity should correlate with known tissue expression levels of NDUFA3

  • Nuclear or diffuse cytoplasmic staining likely indicates non-specific binding

  • Consistent patterns across multiple samples and experimental replicates support specificity

How are NDUFA3 antibodies being utilized in emerging research on mitochondrial dysfunction in neurodegenerative diseases?

NDUFA3 antibodies are becoming valuable tools in neurodegenerative disease research, where mitochondrial dysfunction is increasingly recognized as a key pathological mechanism:

• Alzheimer's Disease Studies:

  • NDUFA3 antibodies facilitate detection of complex I alterations in patient brain tissues

  • Immunoblotting protocols typically use dilutions of 1:500-1:1000 for optimal detection in brain samples

  • Comparison of NDUFA3 levels between affected and unaffected brain regions provides insights into regional vulnerability

• Parkinson's Disease Research:

  • Given the central role of complex I deficiency in Parkinson's, NDUFA3 serves as a marker for monitoring disease progression

  • IHC applications in substantia nigra tissues aid in visualizing mitochondrial changes in dopaminergic neurons

  • Co-localization studies with other complex I subunits help assess complex integrity in disease models

• Methodological Innovations:

  • Multiplexed immunofluorescence combining NDUFA3 with neuronal and glial markers enables cell-type specific analysis

  • Proximity ligation assays using NDUFA3 antibodies help visualize alterations in protein-protein interactions within the complex

  • Live cell imaging applications track dynamic changes in complex I distribution during neurodegenerative processes

• Therapeutic Development Applications:

  • NDUFA3 antibodies enable high-throughput screening of compounds that stabilize complex I

  • Monitoring NDUFA3 levels in response to neuroprotective interventions provides mechanistic insights

  • Assessment of complex I assembly status using native gel electrophoresis followed by NDUFA3 immunoblotting evaluates therapeutic efficacy

As this field advances, researchers should consider validating NDUFA3 antibodies specifically in neural tissues, as the complex I composition and stability may differ from other commonly studied tissues.

What role does NDUFA3 play in protective mechanisms against high glucose-induced cellular damage?

Recent research has revealed NDUFA3's significant role in protecting cells against metabolic stress, particularly in high-glucose conditions:

• Protective Mechanisms:

  • NDUFA3 overexpression counteracts high glucose-induced cellular injuries in human nucleus pulposus cells (HNPCs)

  • This protection operates through several key mechanisms:

    • Suppression of cellular apoptosis

    • Reduction of reactive oxygen species (ROS) production

    • Improvement of mitochondrial membrane potential (MMP)

    • Enhancement of oxygen consumption rate (OCR)

    • Restoration of mitochondrial Complex I activity

• Experimental Evidence:

  • High glucose exposure decreases cell viability, increases apoptotic cells, elevates ROS production, and impairs mitochondrial function in a dose-dependent manner

  • These negative effects are exacerbated by rotenone (a mitochondrial complex I inhibitor) co-treatment

  • NDUFA3 overexpression significantly mitigates these harmful effects

• Transcriptional Regulation:

  • HDAC/H3K27ac mechanisms play crucial roles in regulating NDUFA3 transcription

  • This epigenetic regulation represents a potential therapeutic target for conditions involving mitochondrial dysfunction

• Methodological Approaches:

  • Detection of NDUFA3 expression changes requires sensitive Western blotting techniques (1:500-1:1000 dilution range)

  • Functional assessment should include complementary assays measuring mitochondrial membrane potential, ROS production, and cellular viability

  • The protective effects of NDUFA3 can be reversed using ROS scavengers like N-acetylcysteine, confirming the ROS-dependent mechanism

These findings highlight NDUFA3 as a potential therapeutic target for diabetes-related complications and other conditions characterized by glucose toxicity and mitochondrial dysfunction.

How do mutations in NDUFA3 affect complex I assembly and function in mitochondrial disease models?

Studies on NDUFA3 mutations provide crucial insights into complex I assembly and pathogenesis of mitochondrial diseases:

• Impact on Complex I Assembly:

  • Knockout of NDUFA3 in cultured human cells results in reduced expression of complex I

  • Analysis shows that the complex migrates as a smaller-than-normal-sized entity in the absence of NDUFA3

  • This indicates NDUFA3 plays a critical structural role in the proper assembly and stability of complex I

• Functional Consequences:

  • NDUFA3 mutations may synergize with other complex I subunit mutations to worsen phenotypic expression of mitochondrial diseases

  • These synergistic effects can be detected through:

    • Reduced complex I enzyme activity measurements

    • Altered oxygen consumption rates

    • Increased production of reactive oxygen species

    • Changes in mitochondrial membrane potential

• Experimental Approaches:

  • Blue Native PAGE followed by Western blotting with NDUFA3 antibodies (1:500 dilution) enables visualization of complex I assembly intermediates

  • Proximity ligation assays help map interactions between NDUFA3 and other complex I subunits

  • Immunoprecipitation with NDUFA3 antibodies followed by mass spectrometry identifies interacting partners affected by mutations

  • Live-cell imaging tracks dynamic changes in complex I distribution and mitochondrial morphology

• Therapeutic Implications:

  • Supplementation with wild-type NDUFA3 in deficient cells may restore complex I assembly and function

  • Compounds that stabilize the remaining complex I structure could potentially compensate for NDUFA3 deficiency

  • Targeting downstream consequences (ROS generation, energy deficiency) may alleviate disease phenotypes