NDUFB10 Antibody

What is NDUFB10 and what is its functional significance in mitochondrial research?

NDUFB10 (NADH Dehydrogenase Ubiquinone 1 beta Subcomplex, 10, 22kDa) functions as an accessory subunit involved in the functional assembly of mitochondrial respiratory chain complex I. This protein plays a critical role in the electron transport chain, as Complex I has NADH dehydrogenase activity with ubiquinone as an immediate electron acceptor and mediates the transfer of electrons from NADH to the respiratory chain . NDUFB10 is also known by several alternative names including Complex I-PDSW, NADH-ubiquinone oxidoreductase PDSW subunit, and CI-PDSW . The protein's functional importance is highlighted by recent research demonstrating that genetic variants in NDUFB10 can cause severe mitochondrial disease characterized by complex I deficiency, making it a significant target for both basic and translational research .

What types of NDUFB10 antibodies are available for research applications?

Researchers have access to multiple formats of NDUFB10 antibodies that vary by host, clonality, and validated applications:

When selecting an antibody for your research, consider both the species reactivity requirements and the intended applications. While some antibodies are human-specific, others demonstrate cross-reactivity with rodent and other mammalian models, providing flexibility for comparative studies .

What experimental applications are suitable for NDUFB10 antibodies?

NDUFB10 antibodies have been validated for multiple experimental applications, each requiring specific optimization strategies:

• Western Blotting (WB): The most widely validated application across available antibodies, useful for quantifying total NDUFB10 protein levels and assessing molecular weight .

• Immunohistochemistry (IHC): Both standard and paraffin-embedded (IHC-P) protocols have been validated, allowing tissue-specific localization studies .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Enable subcellular localization studies, particularly valuable for confirming mitochondrial localization patterns .

• Immunoprecipitation (IP): Allows isolation of NDUFB10 and associated proteins for interaction studies or enrichment prior to other analytical techniques .

• Flow Cytometry (intracellular): Permits quantitative analysis of NDUFB10 at the single-cell level .

• ELISA: Provides an alternative quantitative method for NDUFB10 detection in complex samples .

The choice of application should align with your specific research question and the cellular or tissue system being investigated.

What are the recommended protocols for Western blotting with NDUFB10 antibodies?

Successful Western blotting for NDUFB10 requires careful optimization of several parameters:

• Sample preparation: Human cell lines with known NDUFB10 expression (HeLa, HepG2, Jurkat) serve as reliable positive controls .

• Protein loading: 20 μg of total protein per lane has been shown to yield detectable signals in validated protocols .

• Antibody dilution: Optimal dilutions vary by antibody; for example, the recombinant monoclonal antibody EPR16230-47 (ab196019) performs well at 1/10000 dilution in 5% non-fat dry milk (NFDM) in TBST .

• Expected band size: NDUFB10 should appear at approximately 21 kDa, which matches its predicted molecular weight .

• Controls: Include positive controls (e.g., HeLa cell lysate) and negative controls (e.g., isotype control antibody) to confirm specificity.

When analyzing mitochondrial disease samples, be aware that NDUFB10 levels may be significantly reduced or undetectable in patient cells with pathogenic variants, as demonstrated in proteomic studies of patients with intronic NDUFB10 mutations .

How should immunohistochemistry protocols be optimized for NDUFB10 detection?

For robust immunohistochemical detection of NDUFB10, consider these methodological recommendations:

• Tissue preparation: Paraffin-embedded tissues require appropriate section thickness (typically 4-5 μm) and mounting on positively charged slides .

• Antigen retrieval: Heat-mediated antigen retrieval with Tris/EDTA buffer (pH 9.0) is crucial for exposing epitopes that may be masked during fixation .

• Antibody concentration: For antibodies like EPR16230-47, a 1/500 dilution has been validated for human tissues .

• Incubation conditions: Optimize temperature and duration to balance signal strength with background minimization.

• Detection system: HRP-conjugated secondary antibodies (1/500 dilution) followed by DAB chromogen development provides good visualization of NDUFB10 .

• Controls: Include a secondary-only control by substituting PBS for primary antibody to assess non-specific binding .

The expected staining pattern is cytoplasmic, consistent with the mitochondrial localization of NDUFB10. This has been validated in tissues like human transitional cell carcinoma of the bladder .

What considerations are important for immunofluorescence detection of NDUFB10?

Immunofluorescence offers high-resolution visualization of NDUFB10's subcellular distribution, with these key considerations:

• Cell fixation and permeabilization: 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization effectively preserves NDUFB10 epitopes while allowing antibody access .

• Blocking: Thorough blocking is essential to minimize background fluorescence.

• Antibody dilution: A 1/350 dilution of primary antibody (e.g., EPR16230-47) has been validated for immunofluorescence .

• Secondary detection: Fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488-conjugated goat anti-rabbit IgG at 1/500) provide strong visualization .

• Counterstaining: DAPI nuclear counterstain (blue) provides orientation while contrasting with the cytoplasmic mitochondrial staining of NDUFB10 .

The expected pattern is punctate cytoplasmic staining corresponding to mitochondrial networks. Co-staining with established mitochondrial markers can confirm specificity of the localization pattern.

How can researchers address weak or absent NDUFB10 signal in experimental samples?

When facing challenges with NDUFB10 detection, consider these methodological solutions:

• Sample preparation issues:

  • Ensure proper extraction of mitochondrial proteins by using specialized lysis buffers containing detergents suitable for membrane proteins

  • Verify protein integrity by examining housekeeping proteins

  • Consider enriching for mitochondria before analysis to increase target concentration

• Technical optimization:

  • Increase antibody concentration incrementally if signal is weak

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

  • For Western blot, use high-sensitivity detection substrates

  • For immunostaining, optimize antigen retrieval methods

• Biological variables:

  • Consider that true reduction in NDUFB10 levels may occur in certain conditions

  • In mitochondrial disease patients, NDUFB10 may be dramatically reduced (>4-fold reduction) as demonstrated in proteomic analyses

  • Verify results with alternative antibodies targeting different epitopes

• Controls:

  • Always include positive controls (e.g., HeLa cells) known to express detectable levels of NDUFB10

  • Use loading controls appropriate for mitochondrial proteins rather than standard cytosolic markers

Before concluding that NDUFB10 is absent, rule out technical issues through systematic troubleshooting of each experimental step.

How should researchers interpret changes in NDUFB10 expression in disease models?

Proper interpretation of NDUFB10 expression changes requires contextual analysis:

• Comprehensive analysis: Examine NDUFB10 changes in context with other complex I subunits. In patients with NDUFB10 mutations, proteomic analysis revealed decreased abundance of the majority of complex I subunits while other respiratory chain complex subunits remained relatively unchanged .

• Functional correlation: Connect expression changes to functional outcomes. HEK293T cells lacking NDUFB10 exhibit no detectable complex I enzyme activity and severe defects in mitochondrial respiration .

• Quantitative assessment: Use densitometry for Western blots or fluorescence intensity measurements for immunofluorescence, with appropriate normalization to mitochondrial mass markers.

• Disease relevance: Consider the specific role of NDUFB10 in complex I assembly. The pattern of complex I subunit decrease in patient cells with NDUFB10 mutations is consistent with that observed in gene-edited cells lacking NDUFB10, suggesting similar defects in complex assembly .

• Genetic context: In cases with suspected pathogenic variants, investigate both NDUFB10 protein abundance and complex I assembly/function, as even deep intronic variants can cause significant functional defects through aberrant splicing .

These interpretative frameworks help distinguish between primary defects in NDUFB10 and secondary adaptive responses.

How can immunoprecipitation with NDUFB10 antibodies be optimized for interaction studies?

Effective immunoprecipitation of NDUFB10 requires careful attention to several methodological aspects:

• Antibody selection: Choose antibodies specifically validated for immunoprecipitation applications. For example, the rabbit recombinant monoclonal antibody EPR16230-47 has been validated for NDUFB10 immunoprecipitation from human cell extracts .

• Protocol optimization:

  • Use antibody at appropriate concentration (1/50 dilution has been validated for IP)

  • Adjust lysis conditions to preserve protein-protein interactions while effectively solubilizing membrane-associated proteins

  • Consider crosslinking approaches for capturing transient interactions

• Controls:

  • Include input samples (10 μg of whole cell extract) to verify presence of target protein before IP

  • Use isotype control antibodies (e.g., rabbit monoclonal IgG) as negative controls to identify non-specific binding

  • Validate results with reciprocal IPs when investigating potential interacting partners

• Detection methods:

  • For Western blot detection after IP, an antibody dilution of 1/1000 has been validated

  • Consider mass spectrometry analysis of immunoprecipitated material for unbiased identification of interacting proteins

These optimizations are crucial for reliable characterization of NDUFB10's interaction network within the complex I assembly and beyond.

How can NDUFB10 antibodies contribute to multi-omic analysis of mitochondrial disorders?

NDUFB10 antibodies serve as valuable tools in multi-omic approaches to mitochondrial disease research:

• Integrative transcriptomic-proteomic analysis: In a study of mitochondrial disorder patients, RNA sequencing identified aberrant splicing leading to significantly reduced NDUFB10 expression, which was then confirmed at the protein level . This multi-omic approach enabled identification of a deep intronic variant in NDUFB10 that was missed by conventional exome and genome sequencing .

• Correlation of transcript and protein levels: NDUFB10 antibodies can verify whether transcript-level changes translate to altered protein abundance, which is particularly important in mitochondrial disorders where post-transcriptional regulation may be significant.

• Proteome-wide effects: Quantitative proteomic analysis using NDUFB10 antibodies can reveal how defects in this protein affect the broader mitochondrial proteome. In patient fibroblasts with reduced NDUFB10, the majority of complex I subunits showed decreased abundance while other mitochondrial respiratory chain complex subunits remained relatively unchanged .

• Functional correlation: Antibody-based protein quantification can be correlated with functional assays of mitochondrial respiration and complex I activity to establish cause-effect relationships.

This integrated approach has proven essential for elucidating the molecular pathogenesis of mitochondrial disorders associated with NDUFB10 dysfunction.

What role can NDUFB10 antibodies play in studying complex I assembly defects?

NDUFB10 antibodies are instrumental in dissecting complex I assembly mechanisms:

• Assembly intermediate analysis: Combined with Blue Native PAGE (BN-PAGE), NDUFB10 antibodies can track this subunit's incorporation into various assembly intermediates, helping map the sequence and hierarchy of complex I biogenesis.

• Interaction partner identification: Immunoprecipitation with NDUFB10 antibodies followed by mass spectrometry can identify critical interaction partners in the assembly process.

• Subcellular localization: Immunofluorescence with NDUFB10 antibodies can reveal potential assembly microdomains within mitochondria.

• Pathological effects: In patients with NDUFB10 mutations, quantitative proteomic analysis has demonstrated that NDUFB10 deficiency leads to destabilization of complex I in a pattern similar to that observed in gene-edited cells lacking NDUFB10, suggesting specific effects on the assembly process .

• Assembly factor relationships: Co-immunoprecipitation studies can reveal interactions between NDUFB10 and known assembly factors, potentially uncovering new functional relationships.

These applications make NDUFB10 antibodies valuable tools for understanding both the normal assembly process and pathological disruptions in mitochondrial disease scenarios.

How can researchers use NDUFB10 antibodies to evaluate therapeutic interventions for mitochondrial disorders?

NDUFB10 antibodies provide critical readouts for evaluating potential therapeutic strategies:

• Gene therapy assessment: Following introduction of wild-type NDUFB10 via gene therapy approaches, antibodies can quantify restoration of protein expression and localization.

• Splicing modulation: For intronic variants causing aberrant splicing (as identified in patients with NDUFB10-related disease ), NDUFB10 antibodies can measure protein restoration following treatment with splice-modulating oligonucleotides.

• Pharmacological screening: Antibody-based detection methods can serve as readouts in screens for compounds that stabilize complex I or promote proper assembly in the presence of pathogenic NDUFB10 variants.

• Dose-response studies: Quantitative Western blotting with NDUFB10 antibodies allows establishment of dose-response relationships for potential therapeutics.

• Biomarker development: NDUFB10 antibodies can help develop assays for monitoring disease progression and therapeutic response in accessible patient samples.

The specificity and sensitivity of antibody-based detection make these applications particularly valuable for translational research aimed at developing treatments for NDUFB10-associated mitochondrial disorders.

What have recent studies revealed about NDUFB10's role in mitochondrial disease pathogenesis?

Recent research has significantly advanced our understanding of NDUFB10-related pathology:

• Novel genetic mechanisms: Studies have identified deep intronic variants in NDUFB10 that would be missed by conventional exome sequencing. These variants can lead to aberrant splicing, introducing cryptic exons containing premature stop codons that significantly reduce NDUFB10 expression .

• Multi-level evidence: The pathogenicity of NDUFB10 variants has been confirmed through comprehensive analyses:

  • RNA sequencing demonstrated aberrant splicing and reduced expression

  • Protein analysis showed >4-fold reduction in NDUFB10 abundance

  • Quantitative proteomics revealed destabilization of complex I subunits

  • Functional studies indicated severe defects in mitochondrial respiration

• Diagnostic implications: The identification of cryptic splicing defects in NDUFB10 highlights the importance of transcriptomic and proteomic analyses as complementary diagnostic tools when conventional genomic approaches yield uninformative results .

• Mechanism specificity: Studies have shown that NDUFB10 deficiency leads to isolated complex I deficiency, with the majority of complex I subunits showing decreased abundance while other mitochondrial respiratory chain complex subunits remain relatively unchanged .

These findings strengthen the gene-disease association between NDUFB10 and mitochondrial disorders while providing insights into pathogenic mechanisms.

What methodological advances are improving NDUFB10 antibody applications in research?

Several methodological innovations are enhancing the utility of NDUFB10 antibodies:

• Recombinant antibody technology: Development of recombinant monoclonal antibodies like EPR16230-47 offers improved batch-to-batch consistency and specificity compared to traditional antibody production methods .

• Multiplexed detection systems: Integration of NDUFB10 antibodies into multiplexed assays allows simultaneous detection of multiple complex I subunits, providing more comprehensive assessment of assembly states.

• Improved validation approaches: More rigorous validation strategies, including use of knockout controls and cross-platform verification, enhance confidence in antibody specificity.

• Super-resolution microscopy compatibility: Optimization of antibody protocols for super-resolution techniques enables nanoscale visualization of NDUFB10 organization within mitochondrial substructures.

• Quantitative proteomics integration: Combined use of antibody-based enrichment with mass spectrometry allows more comprehensive characterization of NDUFB10 interaction networks and post-translational modifications.

These methodological advances are expanding the research applications of NDUFB10 antibodies beyond traditional approaches, enabling more sophisticated analyses of this important complex I subunit.

What are the emerging applications of NDUFB10 antibodies in broader mitochondrial research?

NDUFB10 antibodies are finding application in several emerging research areas:

• Mitochondrial stress responses: Tracking NDUFB10 levels during various cellular stresses provides insight into how cells regulate complex I assembly and function under adverse conditions.

• Tissue-specific mitochondrial biology: Comparative analysis of NDUFB10 expression across tissues helps understand organ-specific vulnerabilities to mitochondrial dysfunction.

• Aging research: NDUFB10 antibodies are being used to investigate age-related changes in complex I composition and function, a key aspect of mitochondrial theories of aging.

• Metabolic reprogramming: Studies of NDUFB10 in the context of cancer metabolism are revealing how complex I remodeling contributes to metabolic adaptations in malignant cells.

• Mitochondrial-nuclear communication: Investigation of how NDUFB10 deficiency triggers retrograde signaling to the nucleus, potentially influencing nuclear gene expression programs.

These expanding applications demonstrate how NDUFB10 antibodies are contributing to our understanding of mitochondrial biology beyond their traditional role in diagnostics and basic complex I research.