Recombinant Mouse NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 11, mitochondrial (Ndufb11)

What is the basic structure of NDUFB11 protein?

NDUFB11 is a relatively small integral membrane protein (approximately 153 amino acids in humans) that belongs to the "supernumerary" group of subunits in complex I. The protein contains a single transmembrane helical domain (approximately residues 81-109) that is critical for its integration into the mitochondrial membrane. This transmembrane domain contributes significantly to the protein's hydrophobicity and is essential for proper localization and interaction with other mitochondrial membrane proteins . Notably, mutations or deletions within this domain, such as the deletion of phenylalanine 93 (F93del), can lead to shortening of the transmembrane domain and cause rotational rearrangements of succeeding residues in the helix, potentially affecting protein function .

What antibodies are recommended for detecting mouse Ndufb11 in different applications?

Several validated antibodies are available for detecting mouse Ndufb11 across various experimental applications. The R08-7D9 recombinant monoclonal antibody has been genetically engineered to specifically target human NDUFB11 with cross-reactivity to mouse and rat homologs. This antibody has been validated for Western blot (WB) and immunohistochemistry-paraffin (IHC-P) applications . Another option is the rabbit polyclonal antibody A08638-2, which has been validated for multiple applications including Western blot, immunohistochemistry, immunocytochemistry, immunofluorescence, immunoprecipitation, and ELISA with reactivity to human, mouse, and rat NDUFB11 . This antibody has been extensively tested on various mouse tissues including skeletal muscle and heart tissues, detecting NDUFB11 at the expected molecular weight of approximately 18 kDa .

What are the optimal conditions for Western blot detection of mouse Ndufb11?

For optimal Western blot detection of mouse Ndufb11, the following methodology has been validated:

• Sample preparation: Load approximately 30 μg of protein lysate per lane under reducing conditions.

• Electrophoresis: Perform SDS-PAGE on a 12% gel at 80V (stacking gel) followed by 120V (resolving gel) for approximately 2 hours.

• Transfer: Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes.

• Blocking: Block the membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature.

• Primary antibody incubation: Incubate with anti-NDUFB11 antibody at a 1:1000 dilution overnight at 4°C.

• Washing: Wash with TBS-0.1% Tween three times, 5 minutes each.

• Secondary antibody: Probe with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hours at room temperature.

• Detection: Develop signal using an ECL Plus Western Blotting Substrate.

Using this protocol, a specific band for NDUFB11 should be detected at approximately 18 kDa .

How do mutations in NDUFB11 contribute to different disease phenotypes?

Mutations in NDUFB11 have been associated with several distinct clinical phenotypes, highlighting the gene's pleiotropic effects. These include:

• Microphthalmia with linear skin defects (MLS) syndrome: Heterozygous loss-of-function mutations in NDUFB11 (such as c.262C>T and c.402delG) have been identified in patients with this X-linked male-lethal disorder, which is characterized by eye abnormalities and skin defects along with neurological and cardiac abnormalities .

• Histiocytoid cardiomyopathy: Both de novo mutations and X-linked inherited variants have been associated with this condition .

• Sideroblastic anemia: A unique phenotype featuring early-onset sideroblastic anemia without motor or cognitive disabilities has been reported with a de novo mutation in NDUFB11 .

These diverse phenotypes all share an underlying mitochondrial complex I deficiency, but the specific manifestations appear to depend on the exact nature of the mutation and potentially other genetic or environmental factors. This underscores the importance of NDUFB11 in multiple developmental and physiological pathways .

What zebrafish models exist for studying Ndufb11 function?

Zebrafish morphants have been utilized to study NDUFB11 function, particularly in relation to the sideroblastic anemia phenotype. These models were analyzed using o-dianisidine staining (to visualize hemoglobinization) and flow cytometry . The zebrafish model provides an excellent system for studying the effects of NDUFB11 disruption on erythroid development and can be used to assess the consequences of specific mutations. Studies have demonstrated that NDUFB11 deficiency in zebrafish leads to defects in proliferation of erythroid cells, potentially explaining the anemic phenotype observed in patients with certain NDUFB11 mutations .

How can CRISPR/Cas9 be utilized to create disease-relevant NDUFB11 mutations?

CRISPR/Cas9 gene editing has been successfully employed to introduce specific NDUFB11 mutations to study their functional consequences. The methodology involves:

• Design of guide RNA (gRNA) sequences targeting specific regions of NDUFB11

• Transfection of cells with CRISPR/Cas9/GFP plasmid (such as PX458) containing the NDUFB11-targeting guide sequence

• Co-transfection with single-stranded homologous recombination oligo donor sequence containing the desired mutation

• Single clone isolation and screening by restriction digestion (e.g., HinfI digestion) of PCR products spanning the targeted region

• Confirmation by Sanger sequencing

This approach has been utilized to introduce patient-specific knock-in variants, allowing for detailed functional studies of how specific mutations affect NDUFB11 function, complex I assembly, and cellular phenotypes . The approach enables precise modeling of disease-causing mutations and can be applied to various cell types, including stem cells that can be differentiated into relevant lineages.

What are the effective methods for measuring mitochondrial function in NDUFB11-deficient cells?

Several complementary approaches can be used to thoroughly assess mitochondrial function in NDUFB11-deficient cells:

These methods provide complementary information about the consequences of NDUFB11 deficiency on mitochondrial function, from the molecular level (complex assembly) to the functional level (respiration and ATP production).

How can lentiviral transduction be optimized for NDUFB11 rescue experiments?

For successful complementation studies using NDUFB11 lentiviral vectors, the following methodology has proven effective:

• Construct preparation:

  • Amplify full-length NDUFB11 cDNA from a human RNA library using primers containing appropriate restriction sites (e.g., XbaI and XhoI)

  • Subclone the PCR-amplified fragment into a third-generation lentivirus vector such as pcsc-SP-PW (pBOB)

  • Include a control vector expressing GFP to assess transduction efficiency

• Transduction protocol:

  • Establish optimal viral titers for the specific cell type being used

  • Transduce patient and control fibroblasts with lentivirus containing either wild-type NDUFB11 or control vector

  • Allow sufficient time for protein expression (typically 48-72 hours)

• Validation of rescue:

  • Confirm increased NDUFB11 protein levels by Western blot

  • Assess recovery of complex I assembly by blue native gel electrophoresis

  • Measure restoration of complex I activity using in-gel activity assays

When optimized, this approach has demonstrated marked increases in NDUFB11 protein levels and recovery of complex I assembly and activity in patient cells harboring NDUFB11 mutations, confirming the causative role of NDUFB11 deficiency in the observed phenotypes .

What are the critical controls needed when studying NDUFB11 function in cellular models?

When investigating NDUFB11 function in cellular models, several critical controls should be included:

• For gene knockdown or knockout experiments:

  • Non-targeting shRNA or CRISPR controls processed in parallel with experimental samples

  • Rescue experiments with wild-type NDUFB11 to confirm specificity of observed phenotypes

  • Dose-response assessments if using inducible systems to correlate phenotype with degree of knockdown

• For mutation studies:

  • Isogenic wild-type controls that have undergone the same manipulation processes

  • Introduction of multiple different mutations to distinguish mutation-specific effects from general loss-of-function effects

  • Patient cells alongside engineered cell lines to validate relevance of the model

• For functional assessments:

  • Specific inhibitors of complex I (e.g., rotenone) as positive controls for complex I dysfunction

  • Measurements of multiple OXPHOS complexes to distinguish complex I-specific effects from general mitochondrial dysfunction

  • Correlation of biochemical measurements with cellular phenotypes to establish functional relevance

These controls help ensure that observed phenotypes are specifically attributable to NDUFB11 dysfunction rather than experimental artifacts or secondary effects.

What techniques are emerging for studying tissue-specific effects of NDUFB11 deficiency?

Emerging technologies for investigating tissue-specific consequences of NDUFB11 deficiency include:

• Conditional knockout mouse models using tissue-specific Cre recombinase systems to delete Ndufb11 in specific lineages (cardiac, neuronal, hematopoietic, etc.)

• Differentiation of patient-derived or genetically engineered induced pluripotent stem cells (iPSCs) into relevant cell types to study tissue-specific manifestations

• Single-cell transcriptomics and proteomics to identify cell populations most affected by NDUFB11 deficiency within heterogeneous tissues

• Tissue-specific CRISPR activation or repression systems to modulate NDUFB11 expression in specific cell types in vivo

• In vivo imaging techniques to monitor real-time changes in mitochondrial function in animals with NDUFB11 deficiency

These approaches will help address the critical question of why NDUFB11 mutations cause such diverse clinical phenotypes affecting specific tissues despite the protein's ubiquitous expression and seemingly fundamental role in mitochondrial function .

How might NDUFB11 interact with other respiratory complex subunits?

Understanding NDUFB11's interactions with other respiratory complex components represents a frontier in mitochondrial research. Current evidence suggests:

• As a supernumerary subunit, NDUFB11 likely interacts with both core and other accessory subunits of complex I, potentially contributing to complex stabilization

• The transmembrane domain of NDUFB11 (residues 81-109) likely mediates interactions with other membrane-embedded complex I components

• Mutations affecting this domain, such as the F93del mutation, may disrupt these interactions, leading to complex I assembly defects

• NDUFB11 may also have interactions with complex I assembly factors that are not part of the final complex

Advanced techniques including cryogenic electron microscopy (cryo-EM), crosslinking mass spectrometry, and proximity labeling approaches can further elucidate these interactions at the molecular level, potentially identifying therapeutic targets for mitochondrial diseases caused by NDUFB11 deficiency .