NDUFS5 Antibody

What is NDUFS5 and what is its role in cellular function?

NDUFS5 (NADH:Ubiquinone Oxidoreductase Subunit S5) is a member of the NADH dehydrogenase (ubiquinone) iron-sulfur protein family. This 106 amino acid protein functions as an accessory subunit of mitochondrial Complex I (NADH:ubiquinone oxidoreductase), which is the first and largest enzyme complex in the electron transport chain located in the inner mitochondrial membrane. Although NDUFS5 is believed not to be directly involved in catalysis, it plays an essential structural role in Complex I, which transfers electrons from NADH (produced in the Krebs cycle) to ubiquinone, initiating the electron transport chain that ultimately leads to ATP production . NDUFS5 has a calculated molecular weight of approximately 12-15 kDa and is expressed ubiquitously in human tissues, with relatively higher expression in heart, skeletal muscle, liver, kidney, and fetal heart tissues .

How is NDUFS5 related to mitochondrial function and disease?

NDUFS5 is integral to the proper assembly and function of Complex I in the respiratory electron transport chain. Defects or dysfunction in Complex I subunits, including NDUFS5, have significant clinical implications. Research has associated NDUFS5 with several pathological conditions, including Cardiomyopathy, Familial Hypertrophic, 2 and Mitochondrial DNA Depletion Syndrome 9 . Since Complex I is critical for cellular energy production, dysfunction in NDUFS5 can potentially lead to various myopathies and neuropathies characterized by impaired energy metabolism. Understanding NDUFS5's role in these pathways is essential for researchers investigating mitochondrial disorders, neurodegenerative diseases, and metabolic conditions associated with bioenergetic deficiencies .

What types of NDUFS5 antibodies are available for research, and how should I select the appropriate one?

Multiple types of NDUFS5 antibodies are available for research, including polyclonal antibodies that target different epitopes of the protein. Common options include:

• C-terminal targeting antibodies (e.g., ABIN6263573) - rabbit polyclonal antibodies that recognize the C-terminal region of NDUFS5

• Full-length antibodies targeting amino acids 1-106 (e.g., 15224-1-AP) - recognizing the complete NDUFS5 protein

• Center region-specific antibodies - targeting the middle portion of the protein

Selection criteria should include:

• Target species requirements (e.g., human, rat)

• Experimental application needs (WB, IHC, IF, ELISA)

• Antibody clonality preference (polyclonal vs. monoclonal)

• Specific epitope requirements

• Validation data availability

Review the antibody specifications, immunogen sequences, and validation data to ensure the antibody can detect endogenous levels of NDUFS5 in your experimental system .

How can I verify the specificity of NDUFS5 antibodies?

Verification of NDUFS5 antibody specificity requires multiple complementary approaches:

• Western blot validation: Confirm the detection of a single band at approximately 12-15 kDa in various lysates. Compare this with the predicted molecular weight and potential post-translational modifications .

• Knockdown/knockout controls: Test the antibody against cell lysates from NDUFS5-knockdown models (e.g., using shRNA) alongside controls to verify specific band disappearance or reduction, as demonstrated in validation data showing comparison between sh-Control and sh-NDUFS5 transfected HEK-293 cells .

• Cross-reactivity testing: Evaluate the antibody against samples from multiple species if cross-species reactivity is claimed. Available NDUFS5 antibodies have documented reactivity with human and rat samples, with predicted reactivity in other species like pig, zebrafish, and rabbit .

• Immunohistochemical pattern analysis: Compare IHC staining patterns with known NDUFS5 expression profiles in tissues. For example, validation data shows consistent staining in human liver tissue sections at specific dilutions .

• Multiple antibody comparison: When possible, compare results using antibodies targeting different epitopes of NDUFS5 to confirm consistency of findings .

What are the optimal conditions for using NDUFS5 antibodies in Western blotting?

Optimal Western blotting conditions for NDUFS5 antibodies typically include:

For optimal results, include both positive controls (tissues with known high NDUFS5 expression) and negative controls (lysates from NDUFS5 knockdown cells) to confirm specificity, as demonstrated in validation data .

How can NDUFS5 antibodies be effectively used in immunohistochemistry applications?

For effective immunohistochemistry (IHC) applications with NDUFS5 antibodies:

• Sample preparation:

  • For paraffin-embedded tissues: Use standard fixation protocols with formalin or paraformaldehyde

  • Consider antigen retrieval methods (heat-induced epitope retrieval with citrate buffer is common for mitochondrial proteins)

• Antibody conditions:

  • Recommended dilutions range from 1:50 to 1:200, with validation data showing successful staining at 1:100 in human liver tissue

  • Incubation times typically range from 1-2 hours at room temperature or overnight at 4°C

• Detection systems:

  • Both chromogenic (DAB) and fluorescent detection systems are compatible

  • For fluorescent detection, consider using mitochondrial co-markers to confirm subcellular localization

• Controls and interpretation:

  • Include tissues with known high NDUFS5 expression (liver, heart, kidney) as positive controls

  • Expect punctate or reticular cytoplasmic staining consistent with mitochondrial localization

  • Compare staining patterns with mitochondrial distribution markers to confirm specificity

• Quantification approaches:

  • Consider digital image analysis for quantitative assessment of NDUFS5 expression levels

  • When comparing diseased vs. normal tissues, normalize to mitochondrial content markers to account for differences in mitochondrial mass

What are common issues when working with NDUFS5 antibodies and how can they be resolved?

When troubleshooting NDUFS5 antibody applications, remember that as a mitochondrial protein, sample preparation techniques that preserve mitochondrial integrity are crucial. Additionally, because NDUFS5 is relatively small (12-15 kDa), use appropriate gel concentrations (15-20%) in Western blotting for optimal resolution of low molecular weight proteins .

How can NDUFS5 antibodies be used to investigate mitochondrial complex assembly and function?

NDUFS5 antibodies can be powerful tools for investigating mitochondrial Complex I assembly and function through several advanced approaches:

• Blue Native PAGE coupled with Western blotting:

  • This technique allows detection of NDUFS5 within intact Complex I

  • Compare assembly patterns between normal and pathological samples

  • Monitor incorporation of NDUFS5 into subcomplexes during biogenesis

• Co-immunoprecipitation studies:

  • Use NDUFS5 antibodies to pull down interaction partners

  • Identify novel binding partners or assembly factors

  • Compare interactions under normal and stress conditions

• Immunofluorescence co-localization:

  • Combine NDUFS5 antibodies with other Complex I subunit markers

  • Track spatial organization within mitochondria

  • Analyze redistribution under pathological conditions

• Proximity ligation assays (PLA):

  • Detect and quantify interactions between NDUFS5 and other Complex I components

  • Particularly useful for studying assembly dynamics in situ

• Tracking Complex I assembly in disease models:

  • Monitor changes in NDUFS5 incorporation into Complex I in cellular models of mitochondrial disorders

  • Use NDUFS5 antibodies alongside other Complex I subunit antibodies to identify assembly defects

These approaches can provide insights into how NDUFS5 contributes to Complex I structure, assembly, and function in both normal physiology and disease states .

How can NDUFS5 antibodies help investigate mitochondrial dysfunction in disease?

NDUFS5 antibodies offer valuable research tools for investigating mitochondrial dysfunction across multiple disease contexts:

• Quantitative expression analysis:

  • Measure NDUFS5 protein levels in patient samples vs. controls

  • Track changes in NDUFS5 expression in disease progression models

  • Compare expression across different tissues in systemic mitochondrial disorders

• Complex I assembly assessment:

  • Evaluate incorporation of NDUFS5 into Complex I in cardiomyopathy models

  • Identify assembly defects in mitochondrial DNA depletion syndromes

  • Track compensatory changes in related subunits

• Biomarker development:

  • Assess whether NDUFS5 levels correlate with disease severity in mitochondrial disorders

  • Evaluate potential as a diagnostic or prognostic marker

• Therapeutic response monitoring:

  • Track NDUFS5 expression and Complex I assembly in response to experimental therapies

  • Use as a readout for interventions aimed at improving mitochondrial function

• Tissue-specific vulnerability assessment:

  • Compare NDUFS5 expression and incorporation across tissues with different susceptibilities to mitochondrial dysfunction

  • Particularly relevant for neuromuscular and cardiovascular manifestations of mitochondrial disease

What experimental designs are optimal for studying NDUFS5 in the context of neurodegenerative diseases?

For studying NDUFS5 in neurodegenerative disease contexts, consider these optimized experimental designs:

• Patient-derived cellular models:

  • Use NDUFS5 antibodies to analyze protein expression in fibroblasts, induced neurons, or iPSC-derived neural cells from patients with neurodegenerative diseases

  • Compare with age-matched controls to identify disease-specific alterations

• Post-mortem tissue analysis:

  • Apply immunohistochemistry with NDUFS5 antibodies to affected brain regions

  • Combine with markers of neurodegeneration to correlate NDUFS5 alterations with pathology

  • Use recommended IHC dilutions (1:50-1:200) optimized for neural tissues

• Disease progression studies:

  • Establish time-course experiments in animal models of neurodegeneration

  • Track NDUFS5 expression changes relative to disease progression markers

  • Correlate with functional outcomes and mitochondrial integrity measures

• Stress response paradigms:

  • Subject neuronal models to oxidative stress, excitotoxicity, or protein aggregation stressors

  • Monitor NDUFS5 expression, localization, and Complex I incorporation under stress conditions

  • Use Western blotting with recommended dilutions (1:500-1:2000) to quantify changes

• Therapeutic intervention assessment:

  • Test compounds that target mitochondrial function

  • Use NDUFS5 antibodies to assess restoration of Complex I assembly and function

  • Combine with functional readouts of mitochondrial activity

• Multi-modal analytical approach:

  • Integrate NDUFS5 protein analysis with transcriptomic and metabolomic data

  • Construct pathway models that place NDUFS5 alterations in broader context of bioenergetic dysfunction

These approaches can help elucidate how NDUFS5 and Complex I dysfunction contribute to the pathogenesis of neurodegenerative diseases characterized by mitochondrial impairment .