NDUFAF2 Antibody

What is NDUFAF2 and what are its primary cellular functions?

NDUFAF2, also known as mimitin (Myc-induced mitochondrial protein), functions primarily as a molecular chaperone for mitochondrial complex I assembly. Complex I is essential in the electron transfer from NADH to the respiratory chain, with ubiquinone acting as the immediate electron acceptor . NDUFAF2 was originally identified as a myc-controlled gene involved in the proliferation of esophageal carcinoma, and its transcript and protein are upregulated by the proinflammatory cytokine IL-1 . While initially characterized as an assembly factor for Complex I, subsequent research suggests NDUFAF2 assists in proper protein folding within Complex I rather than functioning strictly as an assembly factor . Recent studies have uncovered an unexpected role for NDUFAF2 in primary cilia formation through interaction with centriole protein ARMC9, establishing a novel link between mitochondrial metabolism and ciliary signaling .

What alternative names should researchers be aware of when searching literature on NDUFAF2?

When conducting literature searches, researchers should be aware of multiple nomenclatures for NDUFAF2:

Using these alternative designations in literature searches ensures comprehensive coverage of relevant research across different fields and time periods .

What types of NDUFAF2 antibodies are available for research applications?

Currently, commercial rabbit polyclonal antibodies against NADH dehydrogenase 1 alpha subcomplex assembly factor 2 are available for research applications . These antibodies are typically generated using synthetic peptides derived from human NDUFAF2 as immunogens and are affinity-purified from rabbit antiserum . The antibodies are provided in liquid formulation containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide for stability . For research requiring detection of endogenous NDUFAF2, these antibodies have been validated to detect the protein in human and mouse samples .

What are the recommended applications and dilutions for NDUFAF2 antibodies?

NDUFAF2 antibodies have been validated for specific research applications with recommended dilutions:

For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used as an alternative . Researchers should optimize these conditions for their specific experimental systems.

How should NDUFAF2 antibodies be stored to maintain optimal activity?

For maximum stability and activity, NDUFAF2 antibodies should be stored at -20°C for up to one year from the date of receipt . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency . If frequent use is anticipated, aliquoting the antibody into smaller volumes upon receipt is recommended to minimize freeze-thaw cycles. When working with the antibody, it should be kept cold (on ice or at 4°C) during experimental procedures.

How does NDUFAF2 interact with methionine sulfoxide reductases in mitochondria?

NDUFAF2 has been identified as a binding partner of all four methionine sulfoxide reductases (MSRs) . Using proximity labeling methods (TurboID) to covalently link mitochondrial methionine sulfoxide reductase A (MSRA) to its binding partners in HEK293 cells, researchers identified NDUFAF2 as one of the strongest interaction partners . Co-immunoprecipitation studies confirmed that NDUFAF2 interacts with mitochondrial MSRA in vivo . Both methionine residues in NDUFAF2 (Met85 and Met160) can be oxidized by hydrogen peroxide to methionine sulfoxide and subsequently reduced back to methionine through the combined action of MSRA and MSRB . This oxidation-reduction cycle may represent a regulatory mechanism for NDUFAF2 function or serve as part of an antioxidant defense system within mitochondria .

What experimental approaches have been used to study NDUFAF2's role in complex I assembly?

Researchers have employed multiple experimental approaches to elucidate NDUFAF2's role in complex I assembly:

• Genetic models: Studies in yeast and human cells with NDUFAF2 deficiency have provided evidence for its role as a chaperone for complex I assembly .

• Biochemical assays: Measurements of complex I activity, oxygen consumption rate (OCR), and NAD+/NADH ratio in NDUFAF2-knockout cells have demonstrated the functional impact of NDUFAF2 deficiency on mitochondrial respiration .

• Protein interaction studies: Co-immunoprecipitation and proximity labeling have identified NDUFAF2's interaction partners within the mitochondrial complex I assembly pathway .

• Structural analysis: Transmission electron microscopy has been used to examine potential structural changes in mitochondrial cristae in NDUFAF2-deficient cells, though no significant structural defects were observed despite functional impairments .

How do mutations in NDUFAF2 affect oxidative stress response?

Studies have shown that NDUFAF2 deficiency leads to increased oxidative stress and mitochondrial DNA deletion . The two methionine residues in NDUFAF2 (Met85 and Met160) are susceptible to oxidation by reactive oxygen species, with hydrogen peroxide exposure causing up to 70% oxidation of the protein at 5mM concentration . This oxidation can be reversed by methionine sulfoxide reductases, suggesting a potential role for NDUFAF2 in sensing and responding to oxidative stress .

When examining the individual susceptibility of these methionine residues, researchers found that both M85V and M160V mutants were equally susceptible to oxidation, while the double mutant was resistant . Co-immunoprecipitation of NDUFAF2 and MSRA was equivalent to wild-type with the single mutants but decreased substantially with the double mutant, indicating that these methionine residues are important for the interaction with methionine sulfoxide reductases .

What is the evidence linking NDUFAF2 to primary cilia formation?

Recent research has uncovered a surprising connection between NDUFAF2 and primary cilia formation . Loss of NDUFAF2 in retinal pigment epithelium (RPE) cells results in primary cilia defects, as demonstrated by immunofluorescence staining with polyglutamylated tubulin antibodies . NDUFAF2 was identified as a binding partner for ARMC9, a basal body protein associated with Joubert syndrome, a ciliopathy with defects in the brain, kidney, and eye .

Experimental evidence shows that NDUFAF2 is both necessary and sufficient for cilia formation, with exogenous expression of NDUFAF2 rescuing ciliary defects in cells from patients with known ARMC9 deficiency . This establishes a mechanistic link between mitochondrial metabolism and primary cilia signaling, representing a novel pathway in ciliogenesis.

How does NDUFAF2 mechanistically contribute to ciliogenesis?

NDUFAF2 participates in multiple steps of ciliogenesis through its interaction with centriole proteins . The protein mediates cilia formation by:

• Removing CP110, a key inhibitory protein in ciliogenesis

• Producing ciliary vesicles necessary for cilia formation

• Stabilizing the transition zone at the base of primary cilia

These functions establish NDUFAF2 as a critical factor in the early stages of primary cilia formation, connecting mitochondrial metabolism to this important cellular process. The discovery that a mitochondrial protein like NDUFAF2 plays a direct role in ciliogenesis suggests novel therapeutic approaches for ciliopathies like Joubert syndrome .

What experimental methods are recommended for studying NDUFAF2's role in ciliogenesis?

Based on published research, several experimental approaches are recommended for investigating NDUFAF2's function in ciliogenesis:

• CRISPR-Cas9 gene editing: Generation of NDUFAF2-knockout cell lines, such as in RPE cells, with confirmation by genotyping and Western blot analysis .

• Rescue experiments: Re-expression of wild-type NDUFAF2 in knockout cells to determine if ciliary defects can be reversed, establishing causality .

• Immunofluorescence microscopy: Staining cells with antibodies against ciliary markers (polyglutamylated tubulin) and NDUFAF2 to visualize cilia formation and potential co-localization .

• Protein interaction studies: Co-immunoprecipitation to identify and confirm interactions between NDUFAF2 and centriole proteins like ARMC9 .

• Functional assays: Assessment of ciliary signaling pathways in NDUFAF2-deficient cells to determine the functional consequences of impaired ciliogenesis.

What purification methods are effective for producing recombinant NDUFAF2 protein?

For researchers requiring purified NDUFAF2 protein for biochemical studies, the following purification protocol has been successfully implemented:

• Expression vector construction: His-tagged human NDUFAF2 plasmid constructed in a pETDuet-1 vector using EcoRI and NotI restriction enzymes .

• Protein expression: IPTG-induced expression in bacterial systems, followed by cell lysis and clarification by centrifugation at 21,100g for 30 minutes .

• Affinity purification: Incubation of cleared lysates with Ni-NTA agarose on a rotating platform at 4°C overnight, followed by column loading and washing with buffer containing 50mM Tris (pH 8.0), 300mM NaCl, 20mM imidazole, 0.1mM EDTA, and 1mM PMSF .

• Elution and fractionation: Protein elution with buffer containing 300mM imidazole, followed by collection of 1ml fractions .

• Quality control: Verification of protein purity by gel electrophoresis and Coomassie Blue staining, followed by overnight dialysis against Tris (pH 8.0) with 1mM DTPA at 4°C .

• Mass verification: Determination of exact mass by HPLC-MS to confirm protein identity and integrity .

This protocol has been used successfully for both wild-type NDUFAF2 and methionine-to-valine mutants, enabling comparative biochemical studies .

What approaches are recommended for studying NDUFAF2-protein interactions?

Several complementary approaches have proven effective for investigating NDUFAF2's protein interactions:

• Proximity labeling (TurboID): This method involves creating a fusion protein of NDUFAF2 with TurboID, a biotin ligase that biotinylates proteins in close proximity. After expression in cells and incubation with biotin, proteins interacting with NDUFAF2 can be isolated using streptavidin beads and identified by mass spectrometry .

• Co-immunoprecipitation: For targeted validation of specific interactions, co-transfection of FLAG-tagged NDUFAF2 with potential interaction partners, followed by immunoprecipitation with anti-FLAG antibody and Western blot detection has proven effective .

• Mutational analysis: Generation of point mutations in key residues (such as the methionine residues in NDUFAF2) to determine their importance for protein-protein interactions. This approach revealed that mutation of both methionine residues substantially decreased interaction with MSRA .

• Proteomic analysis: Mass spectrometry-based approaches for identifying post-translational modifications and interaction partners from cell lysates or purified protein complexes .

What quality control measures should be implemented when using NDUFAF2 antibodies?

To ensure reliable results with NDUFAF2 antibodies, the following quality control measures are recommended:

• Validation controls:

  • Positive controls: Use cell lines with known NDUFAF2 expression (e.g., HepG2, SH-SY5Y cells)

  • Negative controls: Include NDUFAF2-knockout cells or samples where the protein is absent

  • Blocking peptide controls: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining

• Antibody characterization:

  • Verify the immunogen sequence used to generate the antibody

  • Test antibody specificity using Western blot to confirm single band at expected molecular weight (approximately 19-20 kDa)

  • For applications beyond Western blot, validate antibody performance in each specific application

• Experimental reproducibility:

  • Use consistent antibody dilutions (WB: 1:500-1:3000; IP: 0.5-4.0μg)

  • Implement standardized protocols for sample preparation and detection methods

  • Include biological replicates to confirm consistency of findings

What are common challenges when detecting NDUFAF2 in Western blots, and how can they be addressed?

Researchers may encounter several challenges when detecting NDUFAF2 by Western blot:

• Low endogenous expression:

  • Problem: Weak signal due to low abundance of NDUFAF2

  • Solution: Increase protein loading (50-100μg total protein), enrich mitochondrial fraction, use higher antibody concentration (1:500 dilution), and employ sensitive chemiluminescence detection systems

• Non-specific bands:

  • Problem: Detection of additional bands besides NDUFAF2

  • Solution: Optimize blocking conditions (5% BSA or milk), increase washing stringency, include NDUFAF2-knockout samples as negative controls, and verify band size (NDUFAF2 should appear at approximately 19-20 kDa)

• Protein degradation:

  • Problem: Multiple smaller bands or smeared signal

  • Solution: Add protease inhibitors during sample preparation, avoid repeated freeze-thaw cycles of samples, maintain cold conditions throughout processing, and use fresh samples when possible

• Antibody specificity issues:

  • Problem: Inconsistent results between different antibody lots

  • Solution: Validate each new antibody lot against a known positive control, consider using antibodies raised against different epitopes of NDUFAF2 for confirmation

What factors should be considered when designing experiments to study NDUFAF2's dual roles in mitochondria and ciliogenesis?

When investigating NDUFAF2's functions in both mitochondria and primary cilia, researchers should consider:

• Cell type selection:

  • Choose cell models that possess primary cilia and have detectable NDUFAF2 expression (e.g., RPE cells)

  • Consider tissue-specific expression patterns when translating findings to in vivo models

• Temporal dynamics:

  • Design time-course experiments to distinguish between immediate and downstream effects of NDUFAF2 manipulation

  • Consider cell cycle synchronization, as ciliogenesis is cell-cycle dependent

• Functional readouts:

  • Include assays for both mitochondrial function (OCR, complex I activity, NAD+/NADH ratio) and ciliary formation/function

  • Use rescue experiments with wild-type and mutant NDUFAF2 to establish causality and separate the two functions

• Technical considerations:

  • When performing immunofluorescence, include co-staining for mitochondrial markers (TOMM20) and ciliary markers (polyglutamylated tubulin)

  • For biochemical assays, consider subcellular fractionation to separate mitochondrial and ciliary/centrosomal pools of NDUFAF2

How can researchers differentiate between direct and indirect effects of NDUFAF2 manipulation?

Distinguishing direct from indirect effects of NDUFAF2 manipulation requires careful experimental design:

By implementing these approaches, researchers can better delineate the direct molecular functions of NDUFAF2 from secondary effects resulting from altered mitochondrial function or impaired ciliogenesis.