uqcc2 Antibody

What is UQCC2 and why is it important in mitochondrial research?

UQCC2 (Ubiquinol-cytochrome-c reductase complex assembly factor 2), also known as C6orf125, MNF1, and Mitochondrial protein M19, is a critical mitochondrial protein required for the assembly of respiratory chain complex III. It plays a vital role in modulating respiratory chain activities including oxygen consumption and ATP production, which consequently affects skeletal muscle differentiation and insulin secretion by pancreatic beta-cells . At the molecular level, UQCC2 is particularly important for cytochrome b translation and stability .

Mutations in UQCC2 have been associated with severe complex III deficiency, leading to conditions such as neonatal encephalomyopathy characterized by intrauterine growth retardation, lactic acidosis, and renal tubular dysfunction . The protein's calculated molecular weight is approximately 15 kDa (126 amino acids), with an observed weight of around 14 kDa in experimental conditions .

What applications are UQCC2 antibodies most commonly used for?

Based on validated research applications, UQCC2 antibodies are most commonly used for:

When using these antibodies, it's recommended to perform titration experiments to determine the optimal antibody concentration for each specific application and experimental setup .

What are the optimal sample types for detecting UQCC2?

UQCC2 has been successfully detected in the following sample types:

• Cell lines: HEK-293 cells and MCF-7 cells show good detection by Western blot and immunofluorescence, respectively .

• Tissue samples: Human liver cancer tissue and breast cancer tissue have shown positive IHC results . Rat spleen, rat heart, and mouse heart have also been validated for IHC applications .

• Mitochondrial fractions: As UQCC2 is localized to the mitochondrial inner membrane and matrix, mitochondrial fractions are excellent sample types for studying this protein .

For optimal IHC results, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 can be used as an alternative .

How can I validate the specificity of a UQCC2 antibody in my experimental system?

Validating UQCC2 antibody specificity is crucial for reliable research outcomes. A comprehensive validation approach includes:

siRNA Knockdown: This is considered a gold standard for antibody validation. Transfect cells with UQCC2-targeting siRNA and compare protein expression to untreated controls by Western blot. A specific antibody will show substantial signal reduction in knockdown samples . When implementing siRNA knockdown:

• Clean your workspace with RNase-decontaminating solutions

• Use RNase-free tips and change gloves frequently

• Run multiple test transfections when working with new cell lines

• Include appropriate negative controls to account for off-target effects

Genetic Models: Fibroblasts from patients with UQCC2 mutations can serve as negative controls, as shown in studies where patient cells exhibited severe complex III deficiency . Complementation by transduction with wild-type UQCC2 restores complex III levels, confirming antibody specificity .

Western Blot Analysis: Detection of a single band at the expected molecular weight (14 kDa) in appropriate cell types, with absence or reduction of this band in knockdown or knockout samples .

What is the relationship between UQCC2 and respiratory chain complex assembly, and how can antibodies help investigate this?

UQCC2 plays a critical role in the assembly of respiratory chain complex III. Research using UQCC2 antibodies has revealed:

• UQCC2 deficiency leads to severe complex III defects with markedly reduced complex III holocomplex levels .

• UQCC2 deficiency affects the stability of its binding partner, UQCC1, which is barely detectable in patient fibroblasts .

• UQCC2 deficiency impacts the levels of complex III subunits, with particular effects on UQCRC1, UQCRC2, and UQCRFS1 .

• Blue Native-PAGE analysis shows that the small amount of complex III detectable in UQCC2-deficient cells is primarily in supercomplex form, with little or no complex III dimer .

To investigate these relationships:

• Perform BN-PAGE analysis with both digitonin and n-dodecyl β-D-maltoside solubilization to examine complex III in both supercomplex and isolated forms .

• Use dual immunodetection methods with antibodies against UQCC2 and complex III subunits (UQCRC2) to study co-localization .

• Combine UQCC2 immunodetection with functional assays such as oxygen consumption or ATP production measurements to correlate protein expression with mitochondrial function .

How should I design experiments to study UQCC2's role in mitochondrial diseases?

When investigating UQCC2's role in mitochondrial diseases such as complex III deficiency, consider the following experimental design elements:

Patient-derived materials: Fibroblasts from patients with UQCC2 mutations show profound complex III deficiency (as low as 5-9% of normal activity) .

Spectrophotometric enzyme assays: These can reveal the impact of UQCC2 deficiency on all respiratory chain complexes. Typically, UQCC2 mutations show:

• Severe complex III deficiency (5-9% residual activity)

• Moderate reduction in complex I (29-37%)

• Moderate reduction in complex IV (51-53%)

• Normal complex II activity

Rescue experiments: Transduction of patient fibroblasts with wild-type UQCC2 can restore complex III activity (from 29% to 73% of control), confirming causality .

Structural analysis: Blue Native-PAGE with both digitonin and n-dodecyl β-D-maltoside solubilization can assess the impact on supercomplexes versus individual complexes .

Protein interaction studies: As UQCC2 interacts with UQCC1, co-immunoprecipitation experiments can help elucidate the molecular mechanisms of complex III assembly defects .

What are the optimal conditions for immunofluorescence detection of UQCC2 in mitochondria?

For optimal immunofluorescence detection of UQCC2 in mitochondria:

• Fixation: Formalin fixation overnight at 4°C has been validated for UQCC2 detection .

• Antigen retrieval: Heat-induced epitope retrieval in 1 mM EDTA, 0.01% Tween-20, pH 8 at 95°C for 45 minutes improves detection .

• Antibody dilution: Use UQCC2 antibody at 1:200-1:800 dilution in antibody diluent with background-reducing components .

• Co-staining strategy: For mitochondrial localization studies, co-stain with:

  • Anti-porin 31HL (1:400) as a mitochondrial outer membrane marker

  • Anti-NDUFS4 (1:100) for complex I visualization

  • Anti-UQCRC2 (1:400) for complex III visualization

• Detection: Use appropriate fluorophore-conjugated secondary antibodies and include DAPI for nuclear counterstaining.

• Confocal microscopy: Image using confocal microscopy with appropriate filter sets for the selected fluorophores.

How do I troubleshoot non-specific binding when using UQCC2 antibodies?

Non-specific binding is a common challenge with antibodies, including those targeting UQCC2. Troubleshooting approaches include:

• Optimal blocking: Use 1% blocking solution in TBS-T for 30 minutes at room temperature before primary antibody incubation .

• Titration of antibody concentration: For each new experimental system, perform a titration series to determine the optimal antibody concentration that provides specific signal with minimal background .

• Validation with knockdown controls: Implement siRNA knockdown of UQCC2 as a negative control to distinguish between specific and non-specific signals .

• Selection of appropriate antibody format: Consider the impact of polyreactivity, which has been linked to specific characteristics including hydrophobicity, charge, and CDR loop flexibility . For challenging applications, choose antibodies that have been validated using rigorous specificity tests.

• Optimization of washing steps: Increase the number and duration of washing steps to reduce non-specific binding. For Western blots and immunofluorescence, at least three 3-minute washes with PBS-T (0.05% Tween-20) are recommended .

How can I optimize Western blot conditions for detecting UQCC2 in different tissue samples?

Optimizing Western blot conditions for UQCC2 detection requires consideration of several factors:

Sample preparation:

• For mitochondrial proteins like UQCC2, isolation of mitochondrial fractions can significantly increase detection sensitivity

• Use 600 μg homogenates for reliable detection

• Include protease inhibitors to prevent degradation

Gel selection:

• Use acrylamide/bisacrylamide gels with appropriate percentage (10-15%) to resolve the relatively small UQCC2 protein (14 kDa)

Transfer conditions:

• Transfer to nitrocellulose membranes for standard applications

• For Blue Native-PAGE, transfer to polyvinylidene difluoride (PVDF) membrane using CAPS buffer (10 mmol/l 3-cyclohexylamino-1-propane sulfonic acid pH 11, 10% methanol)

Antibody dilution and incubation:

• Use UQCC2 antibody at 1:500-1:1000 dilution

• Incubate overnight at 4°C for optimal results

Loading controls:

• Include appropriate loading controls: GAPDH for total protein normalization, porin 31HL for mitochondrial protein normalization

What considerations are important when studying UQCC2 in the context of supercomplexes?

When investigating UQCC2's role in respiratory chain supercomplexes:

• Detergent selection is critical:

  • Digitonin solubilization preserves supercomplex structures

  • n-dodecyl β-D-maltoside solubilization allows visualization of individual complexes

• Blue Native-PAGE conditions:

  • Use 5-13% polyacrylamide gradient gels for effective separation

  • Ensure proper buffer conditions to maintain complex integrity

• Immunodetection strategy:

  • Use antibodies against multiple complex components:

    • NDUFS4 for complex I

    • UQCRC2 (Core 2) for complex III

    • COX2 for complex IV

    • SDHA for complex II

    • ATP synthase subunit α for complex V

• Quantification approaches:

  • Analyze both the individual complex III dimer and the supercomplex forms

  • In UQCC2-deficient samples, the small amount of detectable complex III is primarily in supercomplex form, with restoration of normal distribution after complementation

How does the choice of antibody clone affect UQCC2 detection and experimental outcomes?

The choice between different UQCC2 antibody clones can significantly impact experimental outcomes:

• Polyclonal versus monoclonal considerations:

  • All currently available validated UQCC2 antibodies appear to be polyclonal rabbit antibodies

  • Polyclonal antibodies typically offer higher sensitivity by recognizing multiple epitopes but may show greater lot-to-lot variation

• Immunogen differences:

  • Antibodies generated against different regions of UQCC2 may vary in their detection efficiency

  • Some are generated against fusion proteins (e.g., UQCC2 fusion protein Ag22710)

  • Others use recombinant human UQCC2 protein (amino acids 14-126)

• Application-specific performance:

  • Some antibodies perform better in certain applications:

    • Proteintech 25781-1-AP has been validated in published studies for WB, IHC, and IF

    • Other antibodies may be optimized specifically for IHC (e.g., Elabscience E-AB-65650)

• Cross-reactivity considerations:

  • Antibodies differ in their validated reactivity:

    • Some are validated only for human samples

    • Others show reactivity with human, mouse, and rat samples

How can UQCC2 antibodies contribute to understanding mitochondrial dynamics in disease models?

UQCC2 antibodies can provide valuable insights into mitochondrial dynamics in various disease contexts:

• Complex III deficiency models: UQCC2 antibodies have already proven valuable in diagnosing and characterizing complex III deficiency due to UQCC2 mutations, revealing specific defects in cytochrome b protein expression .

• Study of compensatory mechanisms: In UQCC2-deficient cells, complex IV holoenzyme levels appear increased despite reduced activity, suggesting compensatory mechanisms that can be further explored using UQCC2 antibodies in conjunction with other mitochondrial markers .

• Investigating mitochondrial nucleoid structure: UQCC2 has been identified as a mitochondrial nucleoid factor , and antibodies can help elucidate its role in nucleoid organization and mtDNA maintenance.

• Tissue-specific mitochondrial adaptations: The differential expression and function of UQCC2 across tissues can be investigated using IHC with UQCC2 antibodies to understand tissue-specific responses to mitochondrial dysfunction .

• Therapeutic intervention assessment: UQCC2 antibodies can be used to monitor the restoration of complex III assembly following genetic or pharmacological interventions in disease models.

What are the latest methodological advances in using UQCC2 antibodies for mitochondrial research?

Recent advances in UQCC2 antibody applications include:

• Enhanced validation approaches: Implementation of standardized validation protocols including siRNA knockdown as negative controls has improved antibody reliability . This addresses the reproducibility crisis in biomedical research, where poorly validated antibodies have contributed to an estimated $800 million annual expenditure on low-quality reagents .

• Integration with proteomics: Combining immunoprecipitation with mass spectrometry to identify UQCC2 interaction partners and their dynamics under different physiological conditions.

• Super-resolution microscopy applications: Using UQCC2 antibodies in conjunction with super-resolution microscopy techniques to visualize the precise localization of UQCC2 within mitochondrial substructures.

• Multiplexed imaging approaches: Simultaneous detection of multiple mitochondrial proteins including UQCC2 to understand the spatial relationships between different components of the respiratory chain complexes.

• Combination with functional assays: Integration of UQCC2 immunodetection with live-cell imaging and functional assays to correlate protein expression with mitochondrial dynamics and function.

By leveraging these advanced techniques, researchers can gain deeper insights into the role of UQCC2 in mitochondrial function and disease pathogenesis.