NDUFS1 Antibody

What is NDUFS1 and why is it important in mitochondrial research?

NDUFS1 is a 75 kDa subunit of the NADH:ubiquinone oxidoreductase complex (complex I), serving as a critical component of the mitochondrial electron transport chain. It plays a pivotal role in cellular energy production through oxidative phosphorylation by transferring electrons from NADH to the respiratory chain. NDUFS1 is the largest subunit of complex I and functions as the first iron-sulfur protein to accept electrons from NADH-flavoprotein reductase within the complex .

The importance of NDUFS1 extends beyond energy metabolism to several key cellular processes:

Research on NDUFS1 provides valuable insights into mitochondrial function and diseases associated with mitochondrial dysfunction, making it a crucial target for mitochondrial research .

What are the common applications of NDUFS1 antibodies in research?

NDUFS1 antibodies are versatile tools in mitochondrial research with several validated applications:

Researchers should validate each antibody in their specific experimental system as reactivity and optimal conditions may vary between antibodies from different manufacturers and different sample types .

What species reactivity can be expected with NDUFS1 antibodies?

Most commercially available NDUFS1 antibodies demonstrate cross-reactivity with human, mouse, and rat samples, making them suitable for comparative studies across these species . Some antibodies have also been reported to work with Drosophila samples , although this should be validated for specific applications.

The cross-species reactivity is due to the high conservation of NDUFS1 across mammalian species. When selecting an antibody for your research, consider the following:

• Verify the specific species reactivity claims by the manufacturer

• Check validation data from published literature using the same antibody

• Consider performing a pilot experiment if working with less common species

• For complex I studies across species, consider species-specific differences in complex I structure and assembly

Species-specific positive controls that have been validated for NDUFS1 antibodies include mouse kidney tissue, mouse liver tissue, rat kidney tissue, and human cell lines such as HEK-293, A549, and HeLa cells .

How should NDUFS1 antibodies be validated for specific applications?

Proper validation of NDUFS1 antibodies is crucial for reliable research results. A comprehensive validation approach includes:

• Western blot validation:

  • Use positive controls known to express NDUFS1 (e.g., mitochondria-rich tissues like heart, liver, kidney)

  • Include negative controls such as NDUFS1 knockdown samples or tissues with naturally low expression

  • Verify the expected molecular weight (74-81 kDa)

  • Test antibody specificity by examining single band detection

• Immunoprecipitation validation:

  • Confirm pull-down of the target protein by subsequent western blot

  • Use mouse lung tissue as a positive control for IP applications

  • Include IgG control to assess non-specific binding

• Immunohistochemistry and immunofluorescence validation:

  • Compare staining patterns with published literature

  • Include peptide competition assays to confirm specificity

  • Perform parallel staining with mitochondrial markers to confirm localization

  • Use human liver cancer tissue for IHC validation and HepG2 cells for IF validation

• Knockout/knockdown validation:

  • The most stringent validation method involves testing the antibody in NDUFS1 knockout or knockdown models

  • Several publications have used NDUFS1 knockdown approaches for validation

When reporting validation results, include detailed methodology, antibody catalog numbers, and specific conditions used, as antibody performance can vary between lots and experimental conditions.

What are the recommended protocols for using NDUFS1 antibodies in mitochondrial complex I research?

When studying mitochondrial complex I using NDUFS1 antibodies, consider these methodological approaches:

• For complex I assembly analysis:

  • Blue Native PAGE (BN-PAGE) followed by western blotting with anti-NDUFS1 antibody

  • Sample preparation: Solubilize mitochondrial membranes with dodecyl-β-D-maltoside (DDM) at 0.75% final concentration

  • Purification: Use anion exchange chromatography followed by size exclusion chromatography

  • Load 40 μg of isolated mitochondria for BN-PAGE analysis

• For complex I activity assessment:

  • In-gel activity assays using NADH as substrate and nitrotetrazolium blue (NBT) as the electron acceptor

  • Include both distal and tumor tissues for comparative analysis in cancer research

  • Use densitometric analysis to quantify activity levels

• For respirasome assembly studies:

  • 2D Blue Native/SDS-PAGE to explore levels of subunits integrated into Complex I

  • Use NDUFS1 as a reference for complex I normalization

  • Analyze both nuclear-encoded (NDUFS1, NDUFS3, NDUFB11, NDUFA1) and mitochondrial-encoded (ND1, ND2, ND5) proteins

• For electron transfer analysis:

  • Examine the electron tunneling between iron-sulfur clusters

  • Focus on the transfer between clusters N4 and N5 when studying NDUFS1 mutations

These protocols should be optimized based on your specific research question and experimental system. Always include appropriate controls and standardization methods.

What controls should be included when using NDUFS1 antibodies in experimental designs?

Robust experimental design with appropriate controls is essential for accurate interpretation of results with NDUFS1 antibodies:

• Positive controls:

  • Tissues known to express high levels of NDUFS1 (kidney, liver, heart)

  • Cell lines with confirmed NDUFS1 expression (HEK-293, A549, HeLa cells)

  • Recombinant NDUFS1 protein as a standard for quantitative applications

• Negative controls:

  • NDUFS1 knockdown or knockout samples

  • Secondary antibody-only controls to assess background staining

  • Isotype controls for immunoprecipitation experiments

  • Pre-immune serum controls for polyclonal antibodies

• Loading controls:

  • For mitochondrial fraction analysis: use other mitochondrial proteins like ND5 as loading controls

  • For complex I-specific analysis: use other complex I subunits like NDUFS3 or NDUFV1

  • For whole cell lysates: use common housekeeping proteins like β-actin

• Specificity controls:

  • Peptide competition assays where the antibody is pre-incubated with excess immunizing peptide

  • Use of multiple antibodies targeting different epitopes of NDUFS1

  • For N-terminal vs. C-terminal epitope antibodies, compare detection patterns as demonstrated in studies of ND6 mutations

• Experimental condition controls:

  • Include wild-type and mutant conditions in parallel

  • For disease models, include both affected and unaffected tissues

  • For time-course experiments, include multiple timepoints to capture dynamic changes

Documenting and reporting these controls is essential for result validation and reproducibility.

How can NDUFS1 antibodies be used to study the relationship between complex I dysfunction and disease pathology?

NDUFS1 antibodies have become instrumental in investigating the link between complex I dysfunction and various pathologies:

• Cancer research applications:

  • Use NDUFS1 antibodies to assess expression levels in tumor versus normal tissues

  • Studies have shown opposite prognostic effects of NDUFS1 in different cancers - low expression correlates with poor prognosis in lung cancer while altered expression has been observed in kidney cancer

  • Combine with CD4+ T cell infiltration markers for improved prognostic prediction in renal cell carcinoma

  • Examine NDUFS1 expression in hepatocellular carcinoma to understand mitochondrial dynamics in cancer progression

• Cardiac pathology studies:

  • Investigate NDUFS1 downregulation in cardiac hypertrophy models

  • Use antibodies to track expression changes in response to angiotensin II treatment

  • Combine with markers of mitochondrial dysfunction (MMP, ROS production) to correlate NDUFS1 levels with mitochondrial health

• Neurodegenerative disease research:

  • Examine NDUFS1 protein levels in neurological conditions associated with mitochondrial dysfunction

  • Study the relationship between NDUFS1 mutations and neurodegenerative processes

  • Combine with markers of oxidative stress to understand disease mechanisms

• Renal injury models:

  • Use NDUFS1 antibodies alongside NDUFV1 antibodies to study complex I in renal ischemia-reperfusion injury

  • Examine how complex I subunit expression changes correlate with mitochondrial homeostasis in acute kidney injury

The methodological approach should include comparison between affected and unaffected tissues, correlation with clinical outcomes, and integration with other markers of mitochondrial function.

What insights can NDUFS1 antibodies provide about mitochondrial complex I assembly and stability?

NDUFS1 antibodies are valuable tools for investigating complex I assembly, stability, and dynamics:

• Assembly pathway analysis:

  • Track NDUFS1 incorporation during complex I biogenesis

  • NDUFS1 belongs to the N-module of complex I, which is typically incorporated in later stages of assembly

  • Use 2D Blue Native/SDS-PAGE followed by western blotting with NDUFS1 antibodies to visualize assembly intermediates

• Stability assessment:

  • NDUFS1 mutations can affect the stability of the entire N-module of complex I

  • Compare NDUFS1 levels in complex I between healthy and diseased tissues to assess complex integrity

  • Studies show that even when full Complex I is assembled in some pathological conditions, it may not be as stable as in healthy tissue

• Respirasome formation:

  • NDUFS1 is involved in super-complex formation

  • Analyze higher molecular weight complexes containing NDUFS1 to study respirasome assembly

  • In disease models like hepatocellular carcinoma with ND6 mutations, despite similar NDUFS1 levels, other complex I subunits show lower presence, suggesting compromised stability

• Active vs. deactive conformational states:

  • Complex I exists in active and deactive states with different conformational features

  • While not directly involving NDUFS1, the protein's environment changes between these states

  • Study NDUFS1 interactions with other subunits to understand complex I activity regulation

Methodologically, these studies often combine BN-PAGE, western blotting, immunoprecipitation, and activity assays to provide a comprehensive view of complex I structure and function.

How can researchers use NDUFS1 antibodies to investigate the metabolic reprogramming in disease states?

NDUFS1 antibodies can help uncover the metabolic adaptations that occur in various disease states:

• Cancer metabolism studies:

  • Use NDUFS1 antibodies to track complex I alterations that accompany metabolic shifts in cancer cells

  • Research has shown that complex I dysfunction can drive metabolic reprogramming toward aerobic glycolysis (Warburg effect)

  • Compare NDUFS1 expression between tumor and normal tissues to understand metabolic adaptation

• Metabolomic correlation:

  • Combine NDUFS1 expression analysis with metabolomic profiling

  • Studies of NDUFS1 mutations have shown that despite different proteomic patterns, the metabolic consequences may be similar, such as inhibitory feedback on the TCA cycle and altered glutathione levels

  • Use NDUFS1 antibodies alongside metabolite measurements to establish cause-effect relationships

• Oxidative stress responses:

  • NDUFS1 dysfunction is associated with altered ROS production

  • Use antibodies to correlate NDUFS1 levels with markers of oxidative stress

  • In cardiac hypertrophy models, NDUFS1 knockdown increased mitochondrial ROS production, while overexpression attenuated these effects

• Adaptive responses to complex I inhibition:

  • Study how cells compensate for NDUFS1 deficiency

  • Research has shown that increased expression of NDH2 (type II NADH dehydrogenase) can render complex I dispensable in some organisms

  • Use NDUFS1 antibodies alongside NDH2 markers to understand compensatory mechanisms

Methodologically, these investigations often involve:

• Parallel analysis of multiple complex I subunits

• Integration with functional assays (respiration, ATP production)

• Correlation with metabolic pathway markers

• Consideration of cell type-specific metabolic profiles

What are common technical challenges when using NDUFS1 antibodies and how can they be addressed?

Researchers may encounter several technical issues when working with NDUFS1 antibodies:

• Multiple bands in western blots:

  • Cause: Post-translational modifications, proteolytic cleavage, or non-specific binding

  • Solution: Optimize antibody concentration, use freshly prepared samples, include protease inhibitors during sample preparation, and validate with knockout controls

• Weak or absent signal:

  • Cause: Low NDUFS1 expression, epitope masking, or antibody degradation

  • Solution: Increase antibody concentration, optimize antigen retrieval for IHC (use TE buffer pH 9.0 or citrate buffer pH 6.0), and avoid repeated freeze-thaw cycles of antibody (store at -20°C)

• High background:

  • Cause: Non-specific binding, excessive antibody concentration, or inadequate blocking

  • Solution: Increase blocking time, optimize antibody dilution, include additional washing steps, and use freshly prepared buffers

• Inconsistent results between experiments:

  • Cause: Lot-to-lot variation, sample preparation differences, or protocol inconsistencies

  • Solution: Use the same antibody lot for related experiments, maintain consistent protocols, and include internal controls for normalization

• Discrepancies between different applications:

  • Cause: Epitope accessibility varies between native and denatured conditions

  • Solution: Consider using different antibodies optimized for specific applications, or validate each application independently

• Storage and handling issues:

  • Cause: Antibody degradation due to improper storage

  • Solution: Store according to manufacturer recommendations (typically at -20°C with 0.02% sodium azide and 50% glycerol), avoid repeated freeze-thaw cycles, and consider preparing smaller aliquots

Documentation of these challenges and solutions in your research protocols will help improve reproducibility and troubleshooting efficiency.

How should researchers interpret conflicting data regarding NDUFS1 expression in different disease contexts?

Interpreting contradictory findings regarding NDUFS1 expression requires careful consideration of multiple factors:

• Context-specific regulation:

  • NDUFS1 expression can vary dramatically between disease types and stages

  • In kidney cancer, NDUFS1 has been reported to be increased in one study while decreased in five others

  • In lung cancer, low NDUFS1 expression correlates with poor prognosis

  • Consider the specific disease context, tissue type, and stage when interpreting results

• Methodology differences:

  • Discrepancies may arise from different detection methods (IHC vs. WB vs. RNA-seq)

  • Antibodies targeting different epitopes may yield different results

  • RNA and protein levels may not correlate due to post-transcriptional regulation

  • Standardize methodologies or use multiple complementary approaches

• Sample heterogeneity:

  • Cell type composition varies between samples and may affect NDUFS1 detection

  • Mitochondrial content differs between tissues and pathological states

  • Use cell type-specific markers or single-cell approaches when possible

• Functional versus expression data:

  • NDUFS1 protein levels may not directly correlate with complex I activity

  • Combine expression analysis with functional assays (enzyme activity, respiration)

  • In hepatocellular carcinoma with ND6 mutations, despite similar NDUFS1 levels, complex I activity was significantly reduced

• Compensatory mechanisms:

  • Cells may upregulate other complex I subunits or alternative pathways to compensate for NDUFS1 dysfunction

  • Examine related proteins like NDUFV1 or alternative NADH dehydrogenases

  • Research has shown that high expression of NDH2 can render complex I dispensable

To address these conflicts, researchers should:

• Report comprehensive methodology details

• Include multiple controls and validation approaches

• Consider both expression and functional data

• Acknowledge contradictory findings in the literature

• Propose testable hypotheses to resolve discrepancies

What analytical approaches should be used when correlating NDUFS1 expression with clinical outcomes or phenotypic changes?

When correlating NDUFS1 expression with clinical or phenotypic data, consider these analytical strategies:

• Quantitative analysis of expression:

  • Use standardized scoring systems for IHC (H-score, Allred score)

  • For western blots, normalize to appropriate loading controls

  • For mRNA expression, use validated reference genes

  • Consider both absolute expression and relative changes between conditions

• Statistical approaches:

  • For survival analysis: Use Kaplan-Meier plots with log-rank tests and hazard ratios (HR)

  • For correlation studies: Apply Pearson or Spearman correlation coefficients

  • For comparing groups: Implement appropriate statistical tests (t-tests, ANOVA)

  • Consider multivariate analysis to account for confounding factors

• Combinatorial biomarker strategies:

  • Combine NDUFS1 with other markers for improved prognostic value

  • Research has shown that combining NDUFS1 with CD4+ T cell infiltration provides better prediction in kidney cancer

  • In lung cancer, combining NDUFS1 and NDUFS8 as a panel identified patient groups with up to 14-fold difference in prognosis

• Functional correlation:

  • Integrate NDUFS1 expression data with functional assays

  • Correlate with mitochondrial parameters (membrane potential, ROS production)

  • In cardiac hypertrophy models, NDUFS1 knockdown was correlated with decreased mitochondrial DNA content, reduced membrane potential, and increased ROS

• Mechanistic validation:

  • Use knockdown/overexpression approaches to establish causality

  • Perform rescue experiments to confirm specificity

  • Include time-course analyses to capture dynamic changes

• Data visualization:

  • Use heatmaps to display correlations between multiple parameters

  • Forest plots for meta-analysis of prognostic value across studies

  • Scatter plots with regression lines for continuous variable correlations

These approaches should be tailored to your specific research question, sample size, and data characteristics, with careful consideration of potential confounding factors and biological variability.

What are emerging applications of NDUFS1 antibodies in understanding mitochondrial dynamics and cellular metabolism?

NDUFS1 antibodies are being applied in several cutting-edge research areas that are expanding our understanding of mitochondrial biology:

• Super-resolution microscopy applications:

  • Using fluorescently labeled NDUFS1 antibodies to visualize complex I distribution and dynamics at nanometer resolution

  • Combining with other mitochondrial markers to study respirasome organization in different cellular states

  • Tracking complex I reorganization during mitophagy or mitochondrial fission/fusion events

• Single-cell analysis:

  • Applying NDUFS1 antibodies in mass cytometry or imaging mass cytometry

  • Examining cell-to-cell variability in complex I expression in heterogeneous tissues

  • Correlating NDUFS1 levels with metabolic states at the single-cell level

• Mitochondrial stress responses:

  • Studying how NDUFS1 expression and localization change in response to various stressors

  • Investigating the relationship between NDUFS1 and mitochondrial quality control pathways

  • Research has linked NDUFS1 to mitochondrial membrane potential and ROS production in stress conditions

• Developmental biology:

  • Examining how NDUFS1 and complex I activity during development influences lifespan

  • Research has shown that developmental, but not adult, depletion of complex I shortens lifespan in model organisms

  • Using NDUFS1 antibodies to track complex I assembly during cellular differentiation

• Therapeutic targeting:

  • Monitoring changes in NDUFS1 expression in response to treatments targeting mitochondrial function

  • Developing strategies to modulate NDUFS1 activity for therapeutic purposes

  • Studies in renal ischemia-reperfusion injury suggest that targeting complex I subunits may protect against mitochondrial dysfunction

These emerging applications will benefit from continued improvement in antibody specificity, sensitivity, and adaptation to new technological platforms.

How might NDUFS1 antibodies contribute to understanding the crosstalk between mitochondria and other cellular compartments?

NDUFS1 antibodies are valuable tools for investigating the complex interactions between mitochondria and other cellular structures:

• Mitochondria-nucleus communication:

  • Track NDUFS1 expression changes in response to nuclear signaling

  • Study how nuclear-encoded NDUFS1 coordinates with mitochondrial-encoded complex I subunits

  • Research has shown differential regulation of nuclear versus mitochondrial-encoded subunits in disease states

• Mitochondria-ER contact sites:

  • Use NDUFS1 antibodies alongside ER markers to study functional contacts

  • Investigate how complex I activity influences calcium signaling at these junctions

  • Explore how metabolic stress affects the organization of these contact sites

• Mitochondria and apoptosis pathways:

  • NDUFS1 is a substrate for caspases during apoptosis

  • Use antibodies to track NDUFS1 cleavage as a marker of mitochondrial involvement in cell death

  • Studies have shown that NDUFS1 cleavage is necessary for mitochondrial changes associated with programmed cell death

• Mitochondrial dynamics and quality control:

  • Combine NDUFS1 antibodies with markers of mitophagy to study selective removal of damaged complex I

  • Investigate how complex I deficiency triggers mitochondrial network remodeling

  • Examine the relationship between NDUFS1 expression and mitochondrial biogenesis

• Metabolic signaling hubs:

  • Study how NDUFS1 and complex I interact with metabolic sensors like AMPK

  • Investigate the role of complex I in nutrient sensing and metabolic adaptation

  • Research in cardiac models has linked NDUFS1 to broader metabolic changes beyond mitochondria

These studies often require multiplexed approaches combining NDUFS1 antibodies with other cellular markers and functional assays to provide integrated views of mitochondrial interactions within the cellular ecosystem.

What methodological advances are needed to improve NDUFS1 antibody applications in research?

Several technological and methodological improvements would enhance the utility of NDUFS1 antibodies in research:

• Improved specificity and validation:

  • Development of monoclonal antibodies with epitope mapping to specific domains of NDUFS1

  • Standardized validation protocols across different applications

  • Creation of knockout cell lines as definitive negative controls

  • Implementation of more rigorous reporting standards for antibody validation

• Advanced imaging applications:

  • Development of live-cell compatible NDUFS1 probes to track dynamics in real-time

  • Optimization for super-resolution microscopy techniques

  • Creation of split fluorescent protein tags for studying NDUFS1 interactions in living cells

  • Adaptation for correlative light and electron microscopy to connect function with ultrastructure

• Multiplexing capabilities:

  • Generation of antibodies compatible with multiplexed imaging technologies

  • Development of antibody panels for simultaneous detection of multiple complex I subunits

  • Adaptation for mass cytometry and other high-dimensional single-cell techniques

  • Creation of proximity labeling approaches to study NDUFS1 interaction networks

• Functional antibodies:

  • Development of conformation-specific antibodies that distinguish between active and inactive complex I

  • Creation of antibodies that recognize post-translational modifications of NDUFS1

  • Generation of activity-modulating antibodies for functional studies

  • Production of intrabodies that can track NDUFS1 in living cells

• Quantitative applications:

  • Standardization of quantification methods across laboratories

  • Development of calibrated reference standards for absolute quantification

  • Implementation of digital pathology approaches for automated, objective analysis

  • Creation of internal standard controls for cross-study comparisons

These methodological advances would address current limitations in sensitivity, specificity, and functional analysis, enabling more sophisticated investigations of NDUFS1 biology in health and disease.

NDUFS1 Antibody Applications and Optimization Parameters

Information compiled from multiple sources and represents general guidelines. Specific conditions should be optimized for each experimental system.