NDUFA6 Antibody

What is NDUFA6 and what is its biological function?

NDUFA6 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6) is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). It functions in the transfer of electrons from NADH to the respiratory chain, with ubiquinone believed to be the immediate electron acceptor for the enzyme . Also known as LYR motif-containing protein 6 (LYRM6), Complex I-B14 (CI-B14), or NADH-ubiquinone oxidoreductase B14 subunit, NDUFA6 is a 128 amino acid protein that localizes to the matrix side of the mitochondrial membrane and belongs to the complex I LYR family . Recent research has revealed that NDUFA6 also participates in the regulation of adipogenic differentiation .

What applications can NDUFA6 antibodies be used for?

NDUFA6 antibodies are primarily validated for the following applications:

Most commercial NDUFA6 antibodies show reactivity with human, mouse, and rat samples . For optimal results, it is recommended to titrate the antibody in each testing system as sensitivity may be sample-dependent .

How should NDUFA6 antibodies be stored and handled?

For optimal stability and performance:

• Store at -20°C, where antibodies remain stable for one year after shipment

• For frequent use, short-term storage at 4°C for up to one month is acceptable

• Avoid repeated freeze-thaw cycles as this may compromise antibody integrity

• Most NDUFA6 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting is generally unnecessary for -20°C storage, though some suppliers provide 20μl sizes containing 0.1% BSA for small-scale experiments

How do I select the appropriate NDUFA6 antibody for my research model?

Selection should be based on:

• Species compatibility: Verify reactivity with your experimental model. Most NDUFA6 antibodies react with human, mouse, and rat samples, but cross-reactivity should be confirmed .

• Application suitability: Ensure the antibody is validated for your intended application. For example:

  • For Western blotting: Confirm the antibody detects the expected 15 kDa band

  • For IHC: Check whether antigen retrieval protocols are specified (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0)

• Immunogen consideration: Examine the immunogen used to generate the antibody. Many NDUFA6 antibodies are raised against recombinant fusion proteins or specific amino acid sequences (e.g., amino acids 35-154 of human NDUFA6) . Choosing antibodies targeted to conserved regions may enhance cross-species reactivity.

• Validation in relevant samples: Select antibodies with positive detection in tissues relevant to your research, such as heart tissue for mitochondrial studies or adipose tissue for adipogenic differentiation research .

What controls should I include when using NDUFA6 antibodies?

A robust experimental design should include:

• Positive controls: Include samples known to express NDUFA6, such as:

  • Human, mouse, or rat heart tissue

  • Human cell lines: Raji, HepG2, 22Rv1

  • Mouse kidney and heart tissue

  • Rat kidney tissue

• Negative controls:

  • Primary antibody omission control

  • Isotype control (Rabbit IgG)

  • NDUFA6 knockdown/knockout samples (if available)

• Loading controls: For Western blot, include appropriate housekeeping proteins based on your experimental context.

• Specificity validation: Consider performing peptide competition assays where the antibody is pre-incubated with the immunogen to confirm specificity .

How can I optimize NDUFA6 antibody detection in mitochondrial fractions?

Mitochondrial protein detection requires specific considerations:

• Sample preparation:

  • Use specialized mitochondrial isolation buffers to maintain organelle integrity

  • Consider gentle detergents (0.5-1% digitonin or 1% DDM) for membrane protein solubilization

  • Avoid harsh reducing conditions that may disrupt Complex I structure

• Electrophoresis optimization:

  • Use 10-15% SDS-PAGE gels for optimal resolution of the 15 kDa NDUFA6 protein

  • Consider native PAGE for intact Complex I analysis, where NDUFA6 migrations differs from its monomeric form

• Transfer optimization:

  • Use PVDF membranes with 0.2 μm pore size for small proteins

  • Optimize transfer conditions: 100V for 60 minutes in cold room or 25V overnight

• Signal enhancement:

  • Use high-sensitivity ECL substrates

  • Consider signal amplification systems for low abundance detection

  • Typical working dilutions range from 1:500-1:6000 for Western blot , but titration is recommended

• Background reduction:

  • Extend blocking time (2 hours to overnight)

  • Use 5% BSA instead of milk for phospho-specific detection

  • Include 0.05% Tween-20 in all wash steps

How can I use NDUFA6 antibodies to study Complex I assembly and function?

NDUFA6 antibodies can provide insights into Complex I biology:

• Co-immunoprecipitation approaches:

  • Use cross-linking agents such as disuccinimidyl sulfoxide (DSSO) to capture transient interactions

  • Perform FLAG immunoprecipitations with tagged NDUFA6 constructs to identify binding partners

  • Recent cross-linking mass spectrometry (XL-MS) studies have revealed NDUFA6 interaction with the core CI subunit NDUFS8

• Blue Native PAGE analysis:

  • Monitor NDUFA6 incorporation into assembly intermediates

  • Track changes in Complex I assembly under different experimental conditions

  • Combine with second-dimension SDS-PAGE for subcomplex analysis

• Functional assays:

  • Correlate NDUFA6 protein levels with Complex I enzymatic activity

  • Use spectrophotometric assays to measure NADH dehydrogenase activity in samples with varying NDUFA6 levels

  • Study the relationship between NDUFA6 expression and mitochondrial membrane potential

• Interaction studies:

  • Yeast two-hybrid (Y2H) assays have confirmed specific interaction between NDUFA6 and NDUFS8

  • Mutations in surface patches of NDUFA6 can disrupt this binding interaction

What is known about the relationship between NDUFA6 and adipogenic differentiation?

Recent research has established NDUFA6 as a regulator of adipogenesis:

• Mechanism of action:

  • NDUFA6 regulates adipogenic differentiation via stearoyl-CoA desaturase 1 (Scd1)

  • This pathway represents a potential therapeutic target for obesity treatment

• Experimental approaches:

  • Use transcriptomics and lipidomics approaches to explore NDUFA6 function in adipogenesis

  • Study models include OP9 cells and adipose-derived stem cells

  • Monitor adipogenic markers and lipid accumulation in response to NDUFA6 manipulation

• Therapeutic implications:

  • SCD1 inhibitors demonstrate inhibitory effects on adipogenesis

  • The NDUFA6-SCD1 axis represents a potential intervention point for obesity treatments

How do I troubleshoot non-specific bands in Western blots with NDUFA6 antibodies?

Non-specific binding can be addressed through systematic optimization:

• Antibody specificity verification:

  • Confirm you're using the correct antibody dilution (typically 1:500-1:6000 for WB)

  • Increase antibody dilution if multiple bands appear

  • Consider using a different NDUFA6 antibody targeting a different epitope

• Sample preparation improvements:

  • Ensure complete protein denaturation (heat samples at 95°C for 5 minutes)

  • Add protease inhibitors to prevent degradation products

  • Use fresh samples; avoid repeated freeze-thaw cycles

• Blocking optimization:

  • Increase blocking time or concentration

  • Try alternative blocking agents (5% BSA, 5% milk, commercial blockers)

  • Include 0.1-0.3% Tween-20 in blocking buffer

• Expected results:

  • NDUFA6 typically appears as a single band at approximately 15 kDa

  • Observed molecular weight should match the calculated weight of 15 kDa

What factors might affect NDUFA6 detection in tissue samples?

Several factors can influence NDUFA6 immunodetection in tissues:

• Tissue fixation and processing:

  • Overfixation can mask epitopes; optimize fixation time

  • For IHC, antigen retrieval methods significantly impact results

  • For NDUFA6, TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

• Expression levels by tissue type:

  • NDUFA6 is highly expressed in metabolically active tissues (heart, kidney)

  • Expression may vary with disease state (e.g., lung cancer tissue shows detectable levels)

• Subcellular localization:

  • NDUFA6 localizes to the matrix side of the mitochondrial inner membrane as a peripheral membrane protein

  • Proper permeabilization is essential for antibody access to mitochondrial proteins

• Pathological conditions:

  • Mitochondrial dysfunction in disease states may alter NDUFA6 levels or localization

  • Complex I deficiency disorders may show altered NDUFA6 expression patterns

How can NDUFA6 antibodies be used in gene therapy research?

NDUFA6 gene therapy has shown promising results in certain models:

• Therapeutic potential:

  • Overexpression of NDUFA6 has been used to rescue mitochondrial dysfunction

  • In experimental autoimmune encephalomyelitis (EAE) models, NDUFA6 gene therapy ameliorated neurodegeneration

• Delivery methods:

  • Adeno-associated viral vector serotype 2 (AAV2) has been used successfully to deliver NDUFA6

  • The construct scAAV-NDUFA6Flag has shown efficacy in intravitreal injection models

• Functional outcomes:

  • NDUFA6Flag overexpression rescued retinal complex I activity completely

  • It reduced axonal loss by 73% and retinal ganglion cell loss by 88%

  • RGC apoptosis was reduced by 66%

  • Complex I activity in EAE was restored to normal levels

• Expression verification:

  • NDUFA6 antibodies are essential for confirming transgene expression

  • Immunoprecipitation and blue native PAGE have confirmed integration of exogenous NDUFA6Flag into murine complex I

How can NDUFA6 antibodies contribute to research on mitochondrial diseases?

NDUFA6 antibodies offer valuable tools for mitochondrial disease research:

• Disease-causing variants:

  • Deep mutational scanning approaches have systematically assessed thousands of NDUFA6 genetic variants

  • Experimental data provide functional evidence for over 5,000 NDUFA6 variants

• Complex I assembly studies:

  • NDUFA6 facilitates incorporation of NDUFS8 into Complex I

  • NDUFS8 overexpression can rectify NDUFA6 deficiency, suggesting a therapeutic approach

• Biomarker development:

  • Quantitative analysis of NDUFA6 levels may serve as a biomarker for complex I deficiencies

  • Tissue-specific expression patterns could inform diagnosis and treatment

• Clinical applications:

  • NDUFA6 antibodies provide a clinical resource for aiding diagnosis of NDUFA6-related diseases

  • The antibodies help validate pathogenicity for novel NDUFA6 variants associated with human pathology

What are the latest methodological advances in studying NDUFA6 protein interactions?

Recent technological developments have enhanced our understanding of NDUFA6 interactions:

• Cross-linking mass spectrometry (XL-MS):

  • XL-MS using disuccinimidyl sulfoxide (DSSO) has identified specific interactions between NDUFA6 and NDUFS8

  • This technique captures transient protein-protein interactions that might be missed by conventional approaches

• Deep mutational scanning (DMS):

  • DMS approaches have systematically assessed thousands of NDUFA6 genetic variants

  • Regions of the protein sensitive to mutations suggest functional relevance

  • This approach provides both functional insights and empirical evidence for evaluating variant pathogenicity

• Yeast two-hybrid (Y2H) assays:

  • Y2H has been used to assess physical interactions between NDUFA6 and Q module proteins (NDUFS8, NDUFS7, NDUFAF3)

  • These assays have confirmed the specific interaction between NDUFA6 and NDUFS8

  • Alanine mutations in surface patches highlighted by DMS disrupt binding of NDUFA6 to NDUFS8

• Integrated multi-omics approaches:

  • Combined transcriptomics and lipidomics provide comprehensive insights into NDUFA6 function

  • These approaches have revealed NDUFA6's role in regulating adipogenic differentiation via Scd1