NDUFV3 Antibody

What is NDUFV3 and what is its role in cellular function?

NDUFV3 (NADH dehydrogenase [ubiquinone] flavoprotein 3, mitochondrial) is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). It is also known under several alternative names including CI-9kD, Complex I-9kD, and Renal carcinoma antigen NY-REN-4 . This protein belongs to the complex I NDUFV3 subunit family, with a calculated molecular weight of 12 kDa (108 amino acids), though it is typically observed at approximately 10 kDa in experimental settings .

While NDUFV3 is an integral component of Complex I, current evidence suggests it is not directly involved in the catalytic activity of the complex . Instead, it likely plays structural or regulatory roles in maintaining the integrity and efficiency of the respiratory chain. Recent research has implicated NDUFV3 in the compositional diversity of mammalian respirasomes, suggesting it may have more complex functions than previously understood .

What applications are NDUFV3 antibodies validated for?

NDUFV3 antibodies have been validated for multiple research applications, with specific protocols and dilution recommendations varying by manufacturer. Based on current validation data, these antibodies can be reliably used in:

It's important to note that optimal dilutions may be sample-dependent, and researchers should consider titrating the antibody in their specific experimental system to achieve optimal results . Several publications have successfully utilized NDUFV3 antibodies, particularly in Western blot applications across human and mouse samples .

How do I validate the specificity of NDUFV3 antibodies in my experimental system?

Validating antibody specificity is crucial for reliable results. For NDUFV3 antibodies, consider these validation approaches:

• Positive controls: Include samples known to express NDUFV3, such as HepG2 cells, human heart tissue, or human liver tissue, which have been confirmed to express detectable levels of the protein .

• Molecular weight verification: Confirm that the detected band appears at approximately 10 kDa, which is the observed molecular weight for NDUFV3 .

• Knockdown/knockout validation: Use siRNA or CRISPR to reduce or eliminate NDUFV3 expression, then confirm reduced or absent signal with the antibody.

• Cross-reactivity assessment: Test the antibody on samples from different species if planning cross-species studies. While many NDUFV3 antibodies show primary reactivity with human samples, some have documented reactivity with mouse samples .

• Multiple antibody approach: When possible, compare results using antibodies targeting different epitopes of NDUFV3 to confirm specificity.

What are the known isoforms of NDUFV3 and how can they be distinguished experimentally?

Research has identified alternative isoforms of NDUFV3, including a novel isoform described in recent literature . These isoforms may have distinct subcellular localizations, functions, or tissue-specific expression patterns. When designing experiments to distinguish between NDUFV3 isoforms:

• Isoform-specific antibodies: Determine whether your antibody recognizes all isoforms or is isoform-specific by examining the immunogen sequence. The peptide immunogen used in some antibodies (e.g., KLH-conjugated synthetic peptide encompassing a sequence within the center region of human NDUFV3 ) may recognize multiple isoforms.

• Molecular weight discrimination: Use high-resolution gel electrophoresis systems to separate closely-migrating isoforms that may differ slightly in molecular weight.

• RT-PCR analysis: Complement antibody-based detection with RT-PCR using primers specific to each isoform to confirm isoform expression at the mRNA level.

• Mass spectrometry: For definitive isoform identification, consider immunoprecipitation followed by mass spectrometry analysis to identify peptides unique to specific isoforms.

Recent studies have specifically identified androgen-dependent alternative mRNA isoform expression patterns for NDUFV3 in prostate cancer cells, suggesting potential roles in disease progression .

How does NDUFV3 expression change in pathological conditions?

NDUFV3 expression changes have been documented in several pathological conditions:

• Prostate cancer: Androgens drive changes in both gene and alternative mRNA isoform expression of NDUFV3 in prostate cancer cells. Studies have identified androgen-regulated pathways involving NDUFV3, suggesting a potential role in cancer progression .

• Renal carcinoma: The protein's alternative name (Renal carcinoma antigen NY-REN-4) indicates its association with renal carcinoma, though the precise mechanisms remain under investigation .

• Neurodegenerative disorders: NDUFV3 has been studied in the context of Parkinson's disease due to its role in mitochondrial Complex I. Sequence analysis of nuclear-encoded subunits including NDUFV3 has been performed in Parkinson's disease research .

• Pancreatic cancer: Recent research has implicated NDUFV3 in metabolic mechanisms that may influence pancreatic cancer progression after chemotherapy .

What approaches are recommended for studying NDUFV3's role in mitochondrial complex I assembly?

To investigate NDUFV3's role in Complex I assembly:

• Blue Native PAGE: Use blue native polyacrylamide gel electrophoresis followed by immunoblotting with NDUFV3 antibodies to analyze intact complexes and supercomplexes containing NDUFV3.

• Proximity labeling: Employ BioID or APEX2-based proximity labeling with NDUFV3 as the bait to identify neighboring proteins during complex assembly.

• Pulse-chase experiments: Use metabolic labeling to track the incorporation of newly synthesized NDUFV3 into the complex over time.

• Supercomplex analysis: Recent research on SCAF1's role in driving compositional diversity of mammalian respirasomes has involved NDUFV3 , suggesting its importance in supercomplex formation.

• Quantitative proteomics: Studies have employed quantitative proteomics approaches to reveal oxygen-dependent changes in neuronal mitochondria affecting function and sensitivity to Complex I inhibitors like rotenone, with implications for NDUFV3 function .

When designing such experiments, consider that NDUFV3 is believed to be an accessory subunit rather than a catalytic component, which may influence interpretation of functional outcomes .

What are the optimal sample preparation and antigen retrieval methods for NDUFV3 immunohistochemistry?

For optimal NDUFV3 detection in immunohistochemistry:

• Sample fixation: Standard formalin fixation and paraffin embedding protocols are generally compatible with NDUFV3 detection.

• Antigen retrieval: The preferred method for NDUFV3 antibodies is:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Blocking conditions: Use standard blocking solutions containing serum proteins or commercial blocking reagents compatible with your detection system.

• Antibody dilution: Begin with a dilution range of 1:50-1:500 for IHC applications , optimizing based on your specific tissue and detection method.

• Incubation conditions: Follow manufacturer recommendations, typically overnight incubation at 4°C for primary antibodies.

Positive control tissues with confirmed NDUFV3 expression include human placenta and kidney tissues, which have been validated for IHC applications with NDUFV3 antibodies .

What are the recommended troubleshooting strategies for weak or non-specific signals?

When encountering problems with NDUFV3 antibodies:

• For weak signals:

  • Increase antibody concentration (within recommended ranges)

  • Extend primary antibody incubation time

  • Enhance signal amplification (e.g., biotin-streptavidin systems)

  • Optimize antigen retrieval (try both recommended methods: TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Use fresh reagents and ensure proper storage conditions (-20°C, with glycerol for stability)

• For non-specific signals:

  • Increase blocking stringency

  • Optimize antibody dilution (test dilution series)

  • Reduce primary and secondary antibody incubation times

  • Include additional washing steps

  • Pre-absorb the antibody with the immunizing peptide if available

  • Ensure sample freshness and proper fixation

• For incorrect molecular weight in Western blots:

  • Verify gel percentage is appropriate for resolving 10-12 kDa proteins

  • Ensure appropriate running conditions for small proteins

  • Check for post-translational modifications or processing that might alter migration

What controls are essential when using NDUFV3 antibodies?

Essential controls for NDUFV3 antibody experiments include:

• Positive tissue/cell controls:

  • Western blot: HepG2 cells, human heart tissue, human liver tissue

  • IHC: Human placenta tissue, human kidney tissue

  • IF/ICC: HepG2 cells

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Tissues/cells known not to express NDUFV3

• Specificity controls:

  • Blocking peptide competition (if available)

  • siRNA knockdown samples

  • Genetic knockout samples (when feasible)

• Technical controls:

  • Loading controls for Western blot (e.g., housekeeping proteins)

  • Tissue architecture markers for IHC

  • Subcellular markers for colocalization in IF (particularly mitochondrial markers)

How can I optimize NDUFV3 detection in challenging samples like FFPE tissues?

For challenging samples:

• Extended antigen retrieval: Increase the duration of heat-induced epitope retrieval using TE buffer pH 9.0 .

• Signal amplification: Employ tyramide signal amplification or polymer-based detection systems to enhance sensitivity.

• Dual antigen retrieval: Sequential application of heat and enzymatic antigen retrieval methods.

• Fresh sections: Use freshly cut sections from FFPE blocks rather than stored slides.

• Antibody cocktails: Consider simultaneous application of multiple NDUFV3 antibodies targeting different epitopes.

• Optimized buffers: Add protein carriers or detergents to antibody dilution buffers to reduce background and enhance penetration.

Researchers should note that different tissues may require different optimization strategies, and protocols should be systematically optimized for each tissue type and preparation method.

What are the best practices for quantifying NDUFV3 expression levels?

For accurate quantification of NDUFV3:

• Western blot quantification:

  • Use appropriate loading controls

  • Ensure signal is within linear range of detection

  • Apply standardized normalization methods

  • Consider using fluorescent secondary antibodies for wider linear range

• IHC quantification:

  • Use standardized scoring systems (H-score or Allred)

  • Employ digital image analysis with appropriate thresholding

  • Include intensity calibration standards

  • Consider multiplex IHC for simultaneous analysis of multiple markers

• Fluorescence-based quantification:

  • Use nuclear or cytoskeletal markers for normalization

  • Apply appropriate background subtraction methods

  • Consider subcellular fractionation approaches for compartment-specific quantification

• Avoid common pitfalls:

  • Signal saturation leading to underestimation of differences

  • Non-specific background affecting measurements

  • Improper normalization strategies

  • Failure to account for tissue heterogeneity

How can NDUFV3 antibodies be utilized in studies investigating mitochondrial dysfunction?

NDUFV3 antibodies can provide valuable insights in mitochondrial research:

• Complex I assembly analysis: Track NDUFV3 incorporation as a marker of complex assembly state.

• Oxygen-dependent changes: Recent studies have utilized quantitative proteomics to reveal oxygen-dependent changes in neuronal mitochondria affecting function and sensitivity to rotenone, with NDUFV3 being implicated in these processes .

• Disease models: NDUFV3 antibodies can be used to study changes in neurodegenerative disorders like Parkinson's disease, where mitochondrial dysfunction plays a critical role .

• Cancer metabolism: Investigate NDUFV3's role in metabolic adaptations in cancer, particularly in pancreatic cancer progression after chemotherapy .

• Drug response studies: Assess whether treatments affecting mitochondrial function impact NDUFV3 expression or incorporation into Complex I.

• Respiratory chain supercomplex formation: Explore NDUFV3's involvement in the compositional diversity of mammalian respirasomes, as recent research has implicated this protein in supercomplex assembly .

When designing such studies, researchers should consider complementary approaches such as oxygen consumption measurements, mitochondrial membrane potential assays, and assessment of ROS production to develop a comprehensive understanding of NDUFV3's role.