NDUFV2 Antibody Pair

What is NDUFV2 and why is it important in mitochondrial research?

NDUFV2 (NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa) is a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) that catalyzes electron transfer from NADH through the respiratory chain, using ubiquinone as an electron acceptor. This 24-27 kDa protein contains a binuclear [2Fe-2S] cluster called N1a with a highly conserved binding motif Cys-(X)4-Cys-(X)35-Cys-(X)3-Cys .

NDUFV2 is essential for:

• The catalytic activity of complex I

• Assembly of complex I

• Establishment of the proton gradient across the inner mitochondrial membrane

• Cellular energy metabolism through ATP synthesis

Methodologically, when studying NDUFV2, researchers should recognize it as a nuclear-encoded protein that undergoes mitochondrial targeting, with the cleavage site located around amino acid 32 of the precursor protein, where the first 22 residues function as an efficient mitochondrial targeting sequence .

How is NDUFV2 processed for mitochondrial targeting, and what experimental approaches can verify its localization?

NDUFV2 is synthesized as a precursor protein in the cytosol and must be imported into mitochondria. Research has identified that:

• The cleavage site of NDUFV2 is located around amino acid 32 of the precursor protein

• The first 22 residues of NDUFV2 are sufficient to function as an efficient mitochondrial targeting sequence

• The N-terminus requires a net positive charge and an amphiphilic structure with a balance of basic and hydrophobic amino acids for proper mitochondrial targeting

To experimentally verify NDUFV2 localization, researchers should employ multiple complementary approaches:

• Immunofluorescence microscopy using NDUFV2-specific antibodies and mitochondrial markers

• Biochemical fractionation techniques to isolate mitochondrial, cytosolic, and nuclear fractions followed by Western blot analysis

• Site-directed mutagenesis studies of the N-terminal region to assess mitochondrial import efficiency

For the fractionation approach, researchers should follow protocols similar to those described for isolating mitochondria using kits (e.g., mitochondrial isolation kit for cultured cells). Briefly, cells are collected with PBS, centrifuged at 500×g for 5 min, washed with cold PBS buffer, resuspended with cold lysis buffers A, B, and C, followed by centrifugation at 600×g and then 11,000×g to obtain mitochondrial fractions .

What criteria should be used to select the appropriate NDUFV2 antibody for specific experimental applications?

When selecting NDUFV2 antibodies, researchers should consider multiple factors based on their specific experimental needs:

Additional selection criteria should include:

• Host species (rabbit polyclonal or mouse monoclonal options are available)

• Targeted region (N-terminal, internal, or C-terminal) based on experimental needs

• Cross-reactivity with species of interest (human, mouse, rat, or wider species reactivity)

For sensitive applications requiring specificity verification, knockdown/knockout validation data availability should be considered, with published literature showing successful use in KD/KO systems .

How can researchers validate the specificity of NDUFV2 antibodies in their experimental systems?

A robust validation strategy for NDUFV2 antibodies should include multiple approaches:

• RNA interference validation: Transfect cells with NDUFV2-specific shRNA plasmids and confirm knockdown efficiency by qRT-PCR before antibody testing. For example, validated sequences include:

  • shRNA-1: 5′-CCAGTTGGAAAGTATCACATT-3′

  • shRNA-2: 5′-CCTCCAATGAGAGTATATGAA-3′

  • shRNA-3: 5′-CCATTTGATTTCACACCAGAA-3′

• Western blot validation: Perform side-by-side comparison of control and NDUFV2 knockdown samples, confirming the absence or reduction of the specific 24-27 kDa band. Use β-actin as internal reference.

• Multiple antibody comparison: Test antibodies targeting different epitopes of NDUFV2 (N-terminal, internal region, C-terminal) to confirm consistent detection patterns.

• Species cross-reactivity assessment: If working across species, validate each antibody in multiple species-specific samples to confirm expected cross-reactivity .

For quantitative assessment of knockdown efficiency, Western blot analysis should be performed with densitometry measurements. For instance, specific shRNAs have demonstrated varying knockdown efficiencies: shRNA-2 showed 88.61% downregulation of NDUFV2 protein expression in SMMC-7721/ADR cells compared to control, making it significantly more effective than shRNA-1 (47.10%) and shRNA-3 (53.29%) .

How should researchers design experiments to study NDUFV2 involvement in mitochondrial complex I activity in disease models?

When investigating NDUFV2's role in mitochondrial complex I activity in disease models, researchers should implement a comprehensive experimental design:

• Generate appropriate disease models:

  • For cardiotoxicity studies: Use doxorubicin (DOX) at 4 mg/kg body weight once weekly for 5 weeks (cumulative dose 20 mg/kg) in wildtype and genetically modified mice

  • For cancer studies: Use drug-resistant cell lines (e.g., MCF-7/ADR and SMMC-7721/ADR) with NDUFV2 gene silencing

  • For metabolic studies: Consider sex-specific effects by using both male and female animals

• Mitochondrial isolation and functional assays:

  • Extract mitochondrial fractions using appropriate isolation kits

  • Measure activities of mitochondrial complexes I-V using specific assay kits

  • Perform respirometry analyses on isolated mitochondria using pyruvate/malate as substrate

  • Calculate respiratory control ratio (RCR = state 3/state 4o) and coupling efficiency ([state 3–state 4o]/[state 3–AA])

• Protein interaction studies:

  • Investigate interaction between NDUFV2 and other proteins (e.g., PHB2) using co-immunoprecipitation

  • Confirm interactions with pulldown assays

  • Consider using interactome databases to identify potential interaction partners from the 442 interacting proteins identified for NDUFV2

• Functional consequences assessment:

  • For cardiac function: Use echocardiography to measure parameters including ejection fraction (EF), fractional shortening (FS), and cardiac output (CO)

  • For metabolic studies: Measure respiratory control ratio values on different complex-I substrates

In disease models such as DOX cardiomyopathy, researchers should evaluate how NDUFV2 expression affects mitochondrial complex I activity through interaction with partners like PHB2, which governs the expression of NDUFV2 by promoting its stabilization .

What are the most effective protocols for studying NDUFV2 protein-protein interactions in complex I assembly?

To effectively study NDUFV2 protein-protein interactions in complex I assembly, researchers should employ a multi-technique approach:

• Co-immunoprecipitation (Co-IP):

  • Use 0.5-4.0 μg of NDUFV2 antibody for 1.0-3.0 mg of total protein lysate

  • Extract samples from tissues with known high NDUFV2 expression (e.g., mouse heart tissue)

  • Include appropriate controls (IgG control, input sample)

  • Analyze precipitated proteins by mass spectrometry to identify novel interactors

• Proximity ligation assays (PLA):

  • Visualize protein-protein interactions in situ

  • Use antibody pairs against NDUFV2 and suspected interacting partners

  • Quantify interaction signals in different cellular compartments

• Pull-down assays with recombinant proteins:

  • Express and purify NDUFV2 recombinant protein (available commercial sources use HEK293T expression systems)

  • Use tag-based purification approaches (e.g., C-Myc/DDK-tagged NDUFV2)

  • Verify purity by SDS-PAGE and Coomassie blue staining (>80% purity)

  • Incubate with cell/tissue lysates to pull down interacting partners

• Interactome database analysis:

  • Utilize published data on NDUFV2 interacting proteins (442 documented interacting proteins)

  • Focus on key interactors involved in complex I assembly and function

For example, to study the interaction between PHB2 and NDUFV2 in DOX cardiotoxicity, plasmids encoding wild-type and mutated PHB2 and NDUFV2 can be transfected into HEK293T cells using Lipofectamine 2000 reagent for 48 hours before cell lysis and co-IP experiments. This approach has successfully demonstrated that PHB2 interacts with NDUFV2 to mediate regulatory properties on mitochondrial metabolism .

How can NDUFV2 antibodies be utilized to investigate its role in neurological disorders and cardiovascular diseases?

NDUFV2 antibodies can be strategically employed to investigate its role in both neurological and cardiovascular pathologies:

Neurological disorders applications:

• Parkinson's disease (PD) studies:

  • Use IHC with NDUFV2 antibodies (1:500-1:2000) on brain tissue sections

  • Analyze complex I deficiency in PD models, as NDUFV2 mutations constitute a genetic risk factor and may cause complex I deficiency in this disease

  • Compare NDUFV2 expression patterns in disease vs. control tissues

• Schizophrenia and bipolar disorder:

  • Employ western blotting (1:5000-1:20000) to quantify NDUFV2 expression levels in patient-derived samples

  • Use genetic models with NDUFV2 variants to assess mitochondrial dysfunction

Cardiovascular disease applications:

• Hypertrophic cardiomyopathy:

  • Investigate the deletion mutant (lacking residues 19-40) that exhibits reduced mitochondrial targeting ability

  • Use IF/ICC (1:50-1:500) to assess mitochondrial localization of mutant vs. wild-type NDUFV2

  • Perform mitochondrial fractionation followed by western blotting to quantify mitochondrial vs. cytosolic distribution

• Doxorubicin-induced cardiomyopathy:

  • Study NDUFV2 interaction with PHB2 through co-IP assays

  • Analyze cardiac tissue sections with IHC after doxorubicin treatment

  • Employ echocardiography to correlate NDUFV2 expression with functional parameters including ejection fraction, fractional shortening, and cardiac output

Research has shown that PHB2 governs NDUFV2 expression by promoting its stabilization, while PHB2 deficiency significantly downregulates NDUFV2 in DOX-challenged hearts. Cardiac overexpression of PHB2 alleviates mitochondrial defects in DOX cardiomyopathy both in vivo and in vitro by protecting NDUFV2 function .

What are the methodological considerations when studying NDUFV2's role in cancer progression and drug resistance?

When investigating NDUFV2's role in cancer progression and drug resistance, researchers should consider several methodological approaches:

• Gene silencing strategies:

  • Select appropriate shRNA sequences for effective NDUFV2 knockdown. In drug-resistant cancer cell lines, the following sequences have been validated:

    • shRNA-1: 5′-CCAGTTGGAAAGTATCACATT-3′

    • shRNA-2: 5′-CCTCCAATGAGAGTATATGAA-3′ (most effective in SMMC-7721/ADR with 68.7% inhibition)

    • shRNA-3: 5′-CCATTTGATTTCACACCAGAA-3′ (most effective in MCF-7/ADR)

  • Transfect cells using appropriate reagents like Lipofectamine™ 2000

• Cell proliferation analysis:

  • Following NDUFV2 gene silencing, monitor cellular proliferation at multiple timepoints (24h, 48h, 72h)

  • Research has demonstrated significant inhibition rates:

    • MCF-7/ADR: 67.31% (24h), 73.02% (48h), 69.76% (72h)

    • SMMC-7721/ADR: 68.89% (24h), 71.97% (48h), 74.40% (72h)

• Expression analysis in tumor vs. normal tissues:

  • Use IHC with NDUFV2 antibodies (1:500-1:2000) on tissue microarrays

  • Include positive controls like human prostate cancer tissue with appropriate antigen retrieval methods (TE buffer pH 9.0)

• Mitochondrial function assessment:

  • Isolate mitochondria from control and NDUFV2-manipulated cancer cells

  • Measure respiratory parameters and complex I activity

  • Assess the impact on cellular bioenergetics and metabolic reprogramming

Research has shown that NDUFV2 gene silencing can effectively inhibit the proliferation of drug-resistant cancer cell lines, suggesting its potential as a therapeutic target. The inhibition rate of SMMC-7721/ADR cell proliferation was positively correlated with time, indicating a sustained effect of NDUFV2 knockdown on cancer cell growth .

How can researchers resolve common technical issues when using NDUFV2 antibodies in Western blotting and immunohistochemistry?

When encountering technical challenges with NDUFV2 antibodies, researchers should implement the following optimization strategies:

Western Blotting Troubleshooting:

For NDUFV2 specifically, researchers should note that the observed molecular weight is 24-27 kDa, which may differ slightly from the calculated 27 kDa due to post-translational modifications and processing .

Immunohistochemistry Optimization:

• Antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative approach: Citrate buffer pH 6.0

  • Optimize heating time and temperature based on tissue type

• Antibody incubation conditions:

  • Start with 1:500 dilution and adjust as needed

  • Incubate overnight at 4°C for optimal results

  • For paraffin-embedded sections, ensure complete deparaffinization and rehydration

• Signal detection systems:

  • For low expression tissues, use signal amplification systems

  • For dual-labeling experiments, carefully select compatible detection systems

• Positive and negative controls:

  • Always include known positive tissues (e.g., human prostate cancer tissue, mouse brain tissue, mouse heart tissue)

  • Use isotype controls and secondary-only controls to assess non-specific binding

What approaches can address challenges in detecting low-abundance NDUFV2 in specific tissues or subcellular compartments?

For detecting low-abundance NDUFV2 in challenging samples, researchers should implement sensitivity-enhancing techniques:

• Sample enrichment strategies:

  • Perform subcellular fractionation to isolate mitochondria before analysis

  • Use the protocol described in the literature: centrifuge cells at 500×g, wash with cold PBS, resuspend with lysis buffers, followed by differential centrifugation (600×g and 11,000×g) to obtain mitochondrial fractions

  • Concentrate protein samples using appropriate concentration methods

• Signal amplification techniques:

  • For Western blot: Use enhanced chemiluminescence (ECL) substrates designed for low-abundance proteins

  • For IHC/IF: Implement tyramide signal amplification (TSA) to enhance detection sensitivity

  • For very low expression: Consider proximity ligation assay (PLA) which provides single-molecule detection sensitivity

• Alternative detection approaches:

  • Mass spectrometry-based targeted proteomics (SRM/MRM) for quantitative detection

  • RNA-based methods (in situ hybridization, qRT-PCR) as complementary approaches

  • Consider using highly sensitive monoclonal antibodies with optimized epitope recognition

• Optimized immunoprecipitation protocols:

  • Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

  • Increase incubation time (overnight at 4°C)

  • Use protein A/G magnetic beads for efficient capture

  • Elute with non-reducing conditions to maintain antibody integrity

• Special considerations for NDUFV2 detection:

  • Target tissues with known high expression (heart tissue, brain tissue)

  • For visualizing mitochondrial localization, combine with established mitochondrial markers

  • When studying the mitochondrial targeting sequence variants, careful fixation is critical to preserve the native localization

How can researchers utilize NDUFV2 antibodies to investigate sex-specific differences in metabolism and obesity?

Recent research has revealed important sex-specific regulation of NDUFV2 in metabolism and obesity, requiring specific methodological approaches:

• Sex-specific experimental design requirements:

  • Always include both male and female animals/samples in study design

  • Analyze data separately by sex before pooling

  • Control for hormonal status in female animals (estrous cycle stage)

  • Consider gonadectomy experiments to evaluate the role of sex hormones

• Adipose tissue analysis protocols:

  • For adipose NDUFV2 expression studies, collect both gonadal white adipose tissue (gWAT) and other fat depots

  • Process samples immediately to preserve RNA and protein integrity

  • Employ immunohistochemistry to detect tissue-specific expression patterns

• Sex hormone response elements investigation:

  • Analyze estrogen response elements (EREs) in the regulatory regions of NDUFV2

  • Research has identified specific SNPs (e.g., rs3713670) where alternative sequences match ERE motifs and score higher than reference sequences

  • Compare genotype differences in tissue weight (e.g., gWAT weights) between male and female cohorts

• Mitochondrial function assessment:

  • Measure respiratory control ratio (RCR) and coupling efficiency in isolated mitochondria from WAT using pyruvate/malate as substrate

  • Research has shown that increased NDUFV2 expression resulted in:

    • Increased RCR (state 3/state 4o)

    • Increased coupling efficiency ([state 3–state 4o]/[state 3–AA])

    • These effects were more pronounced in females than males

• Metabolic phenotyping:

  • Measure body composition (fat mass vs. lean mass)

  • Assess plasma glycerol levels as indicators of lipolysis

  • Perform insulin tolerance tests to evaluate insulin sensitivity

  • Data has shown that females overexpressing NDUFV2 had reduced basal/fasting glycemia

Research has demonstrated that NDUFV2 overexpression impacts body fat mass and metabolism in a sex-specific manner, with females showing more pronounced effects. These differences are likely mediated through estrogen regulation of NDUFV2 expression .

What are the best methodological approaches for studying NDUFV2's role in mitochondrial targeting and import mechanisms?

To effectively investigate NDUFV2's mitochondrial targeting and import mechanisms, researchers should implement the following methodological approaches: