NDUFS7 Antibody

What is NDUFS7 and why is it significant in mitochondrial research?

NDUFS7 (NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial) is one of the most highly conserved subunits of mitochondrial respiratory chain complex I. Also known as Complex I-20kD, this 20kDa protein plays a central role in the electron transfer process within the mitochondrial respiratory chain . The protein has a calculated molecular weight of 24 kDa based on its 213 amino acid sequence, though it is typically observed at approximately 20 kDa in experimental contexts . NDUFS7 is critical for understanding mitochondrial function because it serves as a key component in the NADH-ubiquinone oxidoreductase complex that catalyzes electron transfer from NADH to ubiquinone . This makes NDUFS7 an important target for investigating mitochondrial dysfunction in various pathological conditions, particularly neurodegenerative and psychiatric disorders.

What species reactivity can be expected with commercially available NDUFS7 antibodies?

Most commercially available NDUFS7 antibodies demonstrate confirmed reactivity with human, mouse, and rat samples . This cross-species reactivity is likely due to the highly conserved nature of the NDUFS7 protein across mammalian species. Some antibodies have additional predicted reactivity with samples from pig, bovine, horse, sheep, and dog models, though these applications may require further validation by individual researchers . When selecting an NDUFS7 antibody for your research, it is advisable to review the manufacturer's validation data for your specific species of interest, particularly if working with less common model organisms.

What are the optimal applications for NDUFS7 antibodies and their recommended dilutions?

NDUFS7 antibodies have been successfully employed across multiple experimental applications with specific dilution requirements for optimal results. Based on validated research applications, NDUFS7 antibodies are primarily utilized in Western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF), immunocytochemistry (ICC), and enzyme-linked immunosorbent assay (ELISA) .

The following dilution recommendations have been established through extensive validation:

It is important to note that optimal dilutions may be sample-dependent and should be determined empirically for each experimental system . For IHC applications specifically, antigen retrieval techniques significantly impact results, with TE buffer (pH 9.0) generally yielding better results than citrate buffer (pH 6.0) .

How should samples be prepared to maximize detection of NDUFS7 in complex tissues like brain?

When working with complex tissues such as brain specimens, proper sample preparation is crucial for specific and sensitive detection of NDUFS7. For subcellular localization studies, mitochondrial fractionation protocols should be employed to enrich for mitochondrial proteins prior to analysis. This is particularly important given that NDUFS7 is primarily localized to mitochondria.

For immunohistochemical or immunofluorescence detection in brain tissues, researchers should consider the following optimization steps:

• Fixation: Use 4% paraformaldehyde for optimal antigen preservation

• Antigen retrieval: Implement heat-induced epitope retrieval with TE buffer (pH 9.0) as the primary method, with citrate buffer (pH 6.0) as an alternative

• Blocking: Use 5% normal serum that matches the species of the secondary antibody

• Primary antibody incubation: Extend to overnight at 4°C to enhance signal strength

• pH considerations: Be aware that tissue pH can significantly affect NDUFS7 detection, as research has demonstrated a correlation between pH and both NDUFS7 expression and complex I activity levels

These protocols have been successfully applied in research examining NDUFS7 expression in postmortem prefrontal cortex samples .

What controls should be included when using NDUFS7 antibodies in experimental workflows?

Implementing appropriate controls is essential for generating reliable and reproducible data with NDUFS7 antibodies. A comprehensive control strategy should include:

• Positive controls: Validated cell lines with confirmed NDUFS7 expression, such as A549 cells, HeLa cells, or human brain tissue lysates

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • NDUFS7 knockout or knockdown samples (where available)

• Loading controls: For Western blotting, mitochondrial markers such as VDAC or COX IV should be used rather than typical housekeeping proteins

• Additional validation measures:

  • Independent antibody validation using a second antibody targeting a different epitope of NDUFS7

  • Peptide competition assays to confirm specificity

  • Molecular weight verification (expected at approximately 20 kDa)

How can researchers address potential cross-reactivity issues with NDUFS7 antibodies?

Despite the high specificity of commercially available NDUFS7 antibodies, cross-reactivity with other mitochondrial proteins remains a potential concern. To address this issue, researchers should:

• Review epitope information: Select antibodies raised against unique regions of NDUFS7. For example, some antibodies target the internal region , while others target amino acids 164-213 .

• Perform validation experiments:

  • Western blot analysis showing a single band at the expected molecular weight (20-24 kDa)

  • Immunoprecipitation followed by mass spectrometry to confirm the identity of pulled-down proteins

  • Comparative analysis using antibodies targeting different epitopes

• Consider sample type specificity: Different tissue types may show varying levels of non-specific binding. Comprehensive validation should be performed for each new tissue type under investigation.

• Blocking optimization: Adjust blocking conditions (increased BSA or serum percentage) to minimize non-specific binding in problematic samples.

When possible, orthogonal methods of detection (such as mRNA quantification) should be employed to corroborate protein expression findings obtained with antibody-based methods.

How can NDUFS7 antibodies be utilized to investigate mitochondrial dysfunction in neuropsychiatric disorders?

NDUFS7 antibodies have proven valuable for investigating the role of mitochondrial dysfunction in neuropsychiatric disorders. Research has demonstrated altered NDUFS7 expression and complex I activity in postmortem prefrontal cortex samples from individuals with bipolar disorder, major depressive disorder, and schizophrenia .

For researchers investigating these connections, several methodological approaches have yielded valuable insights:

• Quantitative expression analysis: Western blotting with NDUFS7 antibodies has revealed significant differences in protein levels between control subjects and those with psychiatric disorders . This approach requires careful normalization to mitochondrial markers rather than typical housekeeping proteins.

• Correlation with functional measures: NDUFS7 expression levels show significant correlation with complex I activity, providing a link between protein expression and functional outcomes. Complex I activity can be measured by monitoring NADH oxidation at 340 nm .

• Oxidative damage assessment: NDUFS7 antibodies can be combined with markers of oxidative damage (protein carbonylation) and nitrosative stress (3-nitrotyrosine levels) to establish connections between mitochondrial protein expression and oxidative/nitrosative damage .

• pH considerations: Researchers should account for tissue pH when interpreting NDUFS7 expression data, as significant correlations exist between tissue pH and both NDUFS7 levels and complex I activity .

What methodological approaches can be used to study the relationship between NDUFS7, complex I activity, and oxidative stress markers?

To effectively investigate the relationship between NDUFS7 expression, complex I activity, and oxidative stress, researchers should implement a comprehensive methodological approach:

• Simultaneous assessment of multiple parameters:

  • NDUFS7 protein levels via Western blotting

  • Complex I enzymatic activity via spectrophotometric assays

  • Protein oxidation via carbonyl content measurement

  • Tyrosine nitration via 3-nitrotyrosine quantification

• Correlation analysis: Apply Pearson correlation testing to determine the strength of relationships between these parameters, as demonstrated in previous research .

• Experimental models: Complement human postmortem studies with cellular models where NDUFS7 expression can be experimentally manipulated through:

  • siRNA knockdown

  • CRISPR/Cas9 gene editing

  • Overexpression systems

• Mitochondrial functional assessment: Integrate NDUFS7 antibody-based detection with broader mitochondrial function assessment, including membrane potential measurements, ATP production, and reactive oxygen species generation.

What are the most common technical challenges when using NDUFS7 antibodies, and how can they be addressed?

Researchers working with NDUFS7 antibodies may encounter several technical challenges that can affect experimental outcomes. Here are the most common issues and their solutions:

• Low signal intensity:

  • Increase antibody concentration within recommended ranges

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize antigen retrieval methods, using TE buffer (pH 9.0) for IHC applications

  • Enhance detection systems (HRP-conjugated polymers for IHC, high-sensitivity ECL for Western blotting)

• High background signal:

  • Implement more stringent washing procedures

  • Optimize blocking conditions (5% BSA instead of milk for phosphorylated targets)

  • Reduce secondary antibody concentration

  • Pre-adsorb antibodies with tissue powder for highly cross-reactive samples

• Inconsistent results between experiments:

  • Standardize lysate preparation procedures

  • Maintain consistent antibody handling (avoid repeated freeze-thaw cycles)

  • Store antibodies as recommended (-20°C with 50% glycerol)

  • Use the same lot of antibody when possible for longitudinal studies

• Multiple bands on Western blot:

  • Verify the expected molecular weight (approximately 20 kDa)

  • Optimize sample preparation to reduce protein degradation

  • Consider using gradient gels for better resolution

  • Include protease inhibitors in all extraction buffers

How should researchers interpret NDUFS7 antibody results in the context of mitochondrial disease research?

When interpreting NDUFS7 antibody results in mitochondrial disease research, several important considerations should be taken into account: