NDUFB1 Antibody

What is NDUFB1 and what is its function in mitochondrial metabolism?

NDUFB1 is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) that is believed not to be directly involved in catalysis. Complex I functions in the transfer of electrons from NADH to the respiratory chain, with ubiquinone believed to be the immediate electron acceptor for the enzyme . NDUFB1 is a small 58 amino acid single-pass membrane protein (approximately 7 kDa) that localizes to the matrix side of the mitochondrial membrane . It is encoded by a gene that maps to human chromosome 14q32.12, which houses over 700 genes and comprises nearly 3.5% of the human genome .

The proper integration of NDUFB1 into Complex I is essential for maintaining optimal mitochondrial function and energy production. While its precise function is still being investigated, it likely plays a structural role in maintaining the integrity of Complex I, which is critical for cellular energy metabolism.

According to multiple sources, the observed molecular weight in Western blotting is consistently around 7 kDa . Some researchers report a calculated molecular weight of 12 kDa, while the observed molecular weight remains 7 kDa . This discrepancy may be attributed to the protein's hydrophobic nature affecting its migration pattern in SDS-PAGE.

Which species show cross-reactivity with commercially available NDUFB1 antibodies?

Most commercially available NDUFB1 antibodies demonstrate reactivity across multiple species, with varying degrees of cross-reactivity:

When selecting an antibody for cross-species applications, researchers should consider sequence homology between species. For example, one antibody (PA5-66629) has reported sequence identity of 36% to mouse and 39% to rat orthologs , which may affect its cross-reactivity efficiency.

How can researchers validate the specificity of NDUFB1 antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For NDUFB1 antibodies, a multi-faceted validation approach is recommended:

• Positive controls: Use cell lines known to express NDUFB1, such as HEK293T, A459, HL-60, or U2OS cells

• Molecular weight verification: Confirm that detected bands appear at the expected molecular weight (~7 kDa)

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to block specific binding

  • For example, antibody A12826-1 uses a synthetic peptide corresponding to human NDUFB1 as immunogen

• Genetic validation: Use NDUFB1 knockout/knockdown samples as negative controls

  • Similar approaches have been used with other Complex I subunits

• Multiple antibody validation: Compare results using antibodies targeting different epitopes

  • Antibodies targeting N-terminal versus full-length NDUFB1 should be compared

• Cross-application validation: Verify antibody performance across multiple applications (WB, IHC, IF)

  • Antibodies like ab317412 have been validated for Western blot and IHC-P applications

Optimizing Western blotting conditions for NDUFB1 detection requires special consideration due to its small size (7 kDa) and membrane-associated nature:

• Sample preparation:

  • Use appropriate lysis buffers containing detergents suitable for membrane proteins

  • Include protease inhibitors to prevent degradation

  • Avoid excessive heating of samples which may cause aggregation

• Gel selection and electrophoresis:

  • Use high percentage (15-20%) acrylamide gels for better resolution of small proteins

  • Consider Tricine-SDS-PAGE systems for improved separation of low molecular weight proteins

• Transfer conditions:

  • Use PVDF membranes which are better suited for small proteins than nitrocellulose

  • Employ semi-dry transfer or wet transfer with careful optimization of transfer time and voltage

  • Consider using specialized transfer buffers with lower methanol concentration for hydrophobic proteins

• Antibody dilutions:

  • Primary antibody dilutions typically range from 1:500 to 1:2,400 for polyclonal antibodies

  • Monoclonal antibodies may require higher dilutions (1:1,000 to 1:50,000)

  • Overnight incubation at 4°C may improve signal quality

• Detection methods:

  • Enhanced chemiluminescence (ECL) or fluorescent detection systems

  • Longer exposure times may be needed due to the small size of the protein

According to published protocols, successful Western blotting has been reported using antibody dilutions of 1:1,000 for monoclonal antibodies like ab317412 and 1:500-1:2,400 for polyclonal antibodies like 16902-1-AP .

What controls should be included when studying NDUFB1 in mitochondrial dysfunction models?

When investigating NDUFB1 in mitochondrial dysfunction models, robust controls are essential for accurate interpretation:

Research on Complex I deficiencies has employed quantitative quadruple immunofluorescent assays to detect deficiencies in patients with mutations affecting nuclear-encoding structural subunits and assembly factors, which could be adapted for NDUFB1 studies .

What methodological approaches are most effective for studying NDUFB1's role in respiratory supercomplex assembly?

Investigating NDUFB1's role in respiratory supercomplex assembly requires specialized techniques:

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

  • Preserves native protein interactions and complex integrity

  • Can be combined with Western blotting to detect NDUFB1 within supercomplexes

  • Anti-NDUFB8, anti-UQCRC1, or anti-COX IV immunoblot bands with high molecular weights can be used to identify complex I-, complex III-, or complex IV-containing supercomplexes

• Complexome profiling:

  • Combines BN-PAGE with mass spectrometry to identify all components of protein complexes

  • Allows quantitative assessment of NDUFB1 incorporation into supercomplexes

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated knockout or knockdown of NDUFB1

  • Site-directed mutagenesis to study specific residues

  • Similar approaches have been used for other Complex I subunits

• Structural biology techniques:

  • Cryo-electron microscopy to visualize supercomplex architecture

  • Cross-linking mass spectrometry to map interaction interfaces

• Proximity labeling methods:

  • BioID or APEX2 fusion proteins to identify proteins in close proximity to NDUFB1

  • Helps map the interaction network within the supercomplex

For researchers investigating supercomplex assembly, careful solubilization of mitochondrial membranes is critical, with mild detergents like digitonin preserving supercomplex integrity better than harsher detergents like n-dodecyl-β-D-maltoside .

How can researchers distinguish between NDUFB1 deficiency and deficiencies in other Complex I subunits?

Distinguishing between deficiencies in different Complex I subunits requires a multi-parameter analytical approach:

• Subunit-specific antibody panels:

  • Use antibodies against multiple Complex I subunits (NDUFB1, NDUFB8, NDUFS1, NDUFS6) to create expression profiles

  • Different patterns of subunit loss can indicate the primary defect

• Molecular weight discrimination:

  • NDUFB1 has a distinct molecular weight (~7 kDa) compared to other subunits like NDUFB8 (~19-22 kDa)

  • This allows differentiation by Western blotting

• Subcomplex analysis:

  • BN-PAGE can reveal specific subcomplexes that accumulate when different subunits are deficient

  • Different assembly defects produce characteristic patterns

• Genetic testing:

  • Sequencing of genes encoding Complex I subunits

  • Patient-derived cells have shown variable levels of NDUFB8 immunoreactivity despite harboring the same genetic variant (p.Trp22Arg in NDUFB3)

• Functional assays:

  • Specific enzymatic activities may be differentially affected

  • Oxygen consumption measurements with substrates that feed different entry points

• Quantitative proteomics:

  • Mass spectrometry-based approaches to quantify all Complex I subunits

  • Can reveal compensatory changes in other subunits

Research has demonstrated that mutations in different Complex I subunits (NDUFB3, NDUFS4, NDUFS6, NDUFS2, NDUFS3) result in variable patterns of complex assembly defects that can be detected using immunohistochemical techniques .

What are the cutting-edge techniques for studying NDUFB1's involvement in mitochondrial disease mechanisms?

Several innovative approaches are being employed to understand NDUFB1's role in mitochondrial pathologies:

• Quantitative quadruple immunofluorescent assay:

  • Allows simultaneous quantification of multiple proteins in tissue sections

  • Has been used to detect Complex I deficiency in patients with mutations affecting nuclear-encoding structural subunits

  • Enables assessment of NDUFB1 levels relative to other mitochondrial markers

• Patient-derived cellular models:

  • Induced pluripotent stem cells (iPSCs) from patients with mitochondrial disorders

  • Differentiation into affected cell types (neurons, muscle, etc.)

  • Enables study of NDUFB1 in disease-relevant cellular contexts

• CRISPR/Cas9 gene editing:

  • Creation of isogenic cell lines with specific NDUFB1 mutations

  • Introduction of patient-specific mutations to study pathogenicity

  • Rescue experiments to confirm causality

• In vivo models:

  • Transgenic mouse models with altered NDUFB1 expression

  • Similar approaches with NDUFAB1 have revealed roles in obesity and insulin resistance

• Multi-omics integration:

  • Combination of proteomics, transcriptomics, and metabolomics

  • Network analysis to understand system-wide effects of NDUFB1 dysfunction

• Live-cell imaging techniques:

  • Real-time visualization of mitochondrial dynamics and function

  • FRET-based sensors to monitor local ATP production, ROS, or calcium

• High-resolution respirometry:

  • Detailed analysis of respiratory function in isolated mitochondria or permeabilized cells

  • Assessment of substrate-specific effects of NDUFB1 deficiency

These advanced techniques provide comprehensive insights into NDUFB1's function and dysfunction in mitochondrial diseases, enabling better understanding of pathological mechanisms and potential therapeutic targets.

How do post-translational modifications affect NDUFB1 detection and function?

Post-translational modifications (PTMs) can significantly impact both NDUFB1 detection and functional properties:

• Impact on antibody detection:

  • PTMs can mask epitopes, leading to false negative results

  • Some antibodies may be PTM-specific, recognizing only modified or unmodified forms

  • The discrepancy between calculated (12 kDa) and observed (7 kDa) molecular weights may be partially due to PTMs

• Common PTMs affecting mitochondrial proteins:

  • Phosphorylation: Can regulate assembly and activity of respiratory complexes

  • Acetylation: Often responsive to metabolic status

  • Ubiquitination: May regulate turnover and quality control

  • Oxidative modifications: Can indicate oxidative stress damage

• PTM detection methods:

  • Mass spectrometry-based approaches

  • PTM-specific antibodies

  • Mobility shift assays

• Functional implications:

  • PTMs may regulate NDUFB1 incorporation into Complex I

  • Could affect interaction with other subunits or assembly factors

  • May respond to cellular stress or metabolic signals

• Experimental considerations:

  • Sample preparation methods can preserve or disrupt different PTMs

  • Phosphatase inhibitors should be included if phosphorylation is of interest

  • Reducing agents can disrupt certain oxidative modifications

While specific information on NDUFB1 PTMs is limited in the search results, approaches used for studying other respiratory complex subunits could be applied to investigate NDUFB1 modifications.

What is the relationship between NDUFB1 and related proteins like NDUFAB1 in metabolic regulation?

Understanding the functional relationships between NDUFB1 and other mitochondrial proteins provides insights into broader metabolic regulation:

• NDUFB1 and NDUFAB1 functions:

  • NDUFB1: Accessory subunit of Complex I, likely plays structural role

  • NDUFAB1: Involved in lipoic acid metabolism and FeS center formation, crucial for mitochondrial function

• Metabolic impact:

  • NDUFAB1 overexpression protects mice against obesity and insulin resistance when challenged with a high-fat diet

  • NDUFAB1 enhances mitochondrial metabolism through multiple mechanisms

  • The relationship suggests Complex I components may broadly influence metabolic regulation

• Molecular mechanisms:

  • NDUFAB1 activates pyruvate dehydrogenase (PDH) in a lipoylation-dependent manner

  • NDUFAB1 promotes assembly of respiratory complexes and supercomplexes

  • NDUFB1 may contribute to these processes through its role in Complex I structure

• Research approaches:

  • Comparative expression analysis of NDUFB1 and NDUFAB1 in metabolic disorders

  • Co-immunoprecipitation to identify physical interactions

  • Functional assays to assess metabolic impact of altering expression levels

• Therapeutic implications:

  • Understanding these relationships could reveal novel mitochondrial targets to prevent obesity and insulin resistance

  • May provide insights into broader metabolic disease mechanisms

Research indicates that mitochondrial proteins like NDUFAB1 are required for systemic glucose homeostasis and insulin signaling, suggesting similar mitochondrial components, potentially including NDUFB1, may have unrecognized roles in metabolic regulation .