UQCR10 Antibody

What is UQCR10 and why is it significant in mitochondrial function?

UQCR10 (also known as Cytochrome b-c1 complex subunit 9) is a component of ubiquinol-cytochrome c oxidoreductase (Complex III) in the mitochondrial electron transport chain. This complex plays a crucial role in oxidative phosphorylation by catalyzing electron transfer from ubiquinol to cytochrome c, while simultaneously facilitating proton translocation across the mitochondrial inner membrane. During the Q cycle process, 2 protons are consumed from the matrix, 4 protons are released into the intermembrane space, and 2 electrons are transferred to cytochrome c, contributing to the electrochemical gradient that drives ATP synthesis .

UQCR10's significance extends beyond energy production. Research has revealed unexpected functions, including a potential role in viral infections such as hepatitis B virus (HBV), suggesting broader physiological importance than previously recognized . The protein is also known by several alternative names: UCRC, HSPC119, Complex III subunit 9, Complex III subunit X, Cytochrome c1 non-heme 7 kDa protein, and Ubiquinol-cytochrome c reductase complex 7.2 kDa protein .

How do researchers validate the specificity of UQCR10 antibodies?

Ensuring antibody specificity is critical when working with UQCR10, particularly due to documented cross-reactivity issues with related mitochondrial proteins. A comprehensive validation strategy includes multiple approaches:

Western Blot Validation:

• Confirm the molecular weight - UQCR10 should appear at approximately 7 kDa

• Include positive controls (e.g., HepG2 cells) known to express UQCR10

• Test the antibody on UQCR10 knockdown/knockout samples as negative controls

Cross-reactivity Assessment:
Research has specifically identified that some anti-UQCRQ antibodies cross-react with UQCR10, which creates potential confusion in experimental interpretation . When evaluating antibody specificity, researchers should:

• Test multiple antibodies targeting different epitopes

• Perform peptide competition assays using the immunizing peptide

• Consider using tagged versions of UQCR10 (such as HA-tagged constructs) to distinguish from endogenous protein and verify specificity

The detection of a band at the expected 7 kDa molecular weight alone is insufficient to confirm antibody specificity, as demonstrated by studies showing that antibodies intended for UQCRQ detection actually recognize UQCR10 instead .

What are the optimal sample preparation techniques for UQCR10 antibody applications?

Effective sample preparation is crucial for successful detection of this small (7 kDa) mitochondrial protein:

For Western Blotting:

• Use RIPA or NP-40 buffer supplemented with protease inhibitors for general cell lysates

• For preserving native protein complexes, consider milder detergents:

  • Digitonin (1-2%) is preferred for maintaining intact respiratory complexes and supercomplexes

  • Lauryl maltoside (1%) provides an intermediate level of solubilization

For Immunoprecipitation:

• Use non-denaturing lysis buffers containing mild detergents

• Pre-clear lysates with Protein A/G beads to reduce non-specific binding

• For co-immunoprecipitation of intact complexes, digitonin (1-2%) is superior to other detergents

For Cell Lines vs. Tissues:
Research has revealed that cell lines may have post-transcriptional inhibition of UQCR10 expression. HepG2 and Huh7 cell lines show normal UQCR10 mRNA levels but significantly reduced protein levels compared to human liver tissues . This finding has important implications for sample preparation:

• Cell lines may require enrichment or concentration steps to detect UQCR10

• Tissue samples generally show higher expression but require careful handling to preserve mitochondrial integrity

• When comparing different sample types, process them in parallel with appropriate normalization controls

What are the validated technical applications for UQCR10 antibodies?

UQCR10 antibodies have been successfully applied in several key experimental techniques:

Western Blotting:

• Successfully detects the 7 kDa UQCR10 protein in multiple cell lines (HepG2, Jurkat, Saos-2, HeLa)

• Optimal dilution ranges from 1:1000 to 1:5000

• Requires appropriate gel percentage (12-15% or gradient gels) to properly resolve this small protein

• PVDF membranes with 0.2 μm pore size are preferred over 0.45 μm for small proteins

Immunoprecipitation:

• Effectively pulls down UQCR10 and its interaction partners

• Typically used at 1:50 dilution for immunoprecipitation applications

• Can be combined with mass spectrometry to identify interaction networks

Complex Composition Analysis:

• Blue Native PAGE combined with western blotting using UQCR10 antibodies can track this protein across different complex assemblies

• Complexome profiling combines BN-PAGE with mass spectrometry to provide comprehensive protein complex mapping

The following table summarizes the validated applications and conditions for UQCR10 antibody use:

How can UQCR10 antibodies be used to study respiratory chain supercomplexes?

Respiratory chain supercomplexes (RCS) represent higher-order assemblies of individual respiratory complexes. UQCR10 antibodies provide valuable tools for investigating these structures:

Blue Native PAGE Combined with Western Blotting:

• Solubilize mitochondria using mild detergents (digitonin is preferred)

• Separate complexes on blue native gels

• Perform Western blotting with UQCR10 antibodies

• Analyze UQCR10 distribution across different molecular weight complexes:

  • Individual Complex III (~500 kDa)

  • Complex III₂+IV (~700 kDa)

  • Complex I+III₂ (~1,500 kDa)

  • Complex I+III₂+IV₁₋₄ (1,700-2,700 kDa)

Complexome Profiling Analysis:
Research has used this technique to analyze the distribution of UQCR10 in different assembly states. When Complex III assembly is impaired (as in cells lacking functional cytochrome b), UQCR10 accumulates in assembly intermediates of various molecular weights ranging from 25 to 2,952 kDa .

For accurate quantification, researchers calculate the area under the peaks defined by relative peptide intensity in complexome profiles, using unrelated mitochondrial proteins (TOM20, TOM22, citrate synthase) as "internal standard" controls .

Investigation of Supercomplex Assembly Factors:
Research has identified that COX7A2L (SCAFI) binds preferentially to Complex III₂ . UQCR10 antibodies can be used in co-immunoprecipitation studies to identify such interactions with assembly factors and study how they change under different conditions.

What methodologies can reveal UQCR10 protein-protein interactions?

Several complementary approaches can identify UQCR10 interaction partners:

Immunoprecipitation with Mass Spectrometry:

• Tag UQCR10 with an epitope tag (such as HA) when using cell models

• Use anti-tag or anti-UQCR10 antibodies for immunopurification

• Identify co-precipitating proteins by mass spectrometry

Research using this approach with SILAC (Stable Isotope Labeling with Amino acids in Cell culture) quantification has revealed that UQCR10 interacts strongly with:

• CYC1 and UQCRH (Complex III components)

• Several Complex IV subunits including MT-CO2, COX5B, COX6C, and COX6B1

• GHITM, CHCHD3 (MIC19, a MICOS complex component), and HADHB

This interaction profile suggests UQCR10 may play a role in both respiratory complex assembly and mitochondrial ultrastructure organization.

How does UQCR10 contribute to HBV infection, and how can antibodies help study this mechanism?

A surprising connection between UQCR10 and hepatitis B virus (HBV) infection has been discovered through research using a transgenic HBV model:

Key Research Findings:

• HepG2 and Huh7 cell lines, which typically resist sustained HBV infection, show normal UQCR10 mRNA levels but significantly reduced protein levels, suggesting post-transcriptional inhibition .

• Restoration of UQCR10 protein expression enables these cell lines to support sustained infection by HBV virions .

• In experiments with cell populations having variable UQCR10 expression, HBV preferentially establishes persistent replication in cells with higher UQCR10 levels .

• The viral entry process involving UQCR10 is dependent on the viral preS1 protein, as blocking with preS1 antibodies strongly inhibits infection .

Experimental Approaches Using UQCR10 Antibodies:

• Western blotting to quantify UQCR10 protein levels in different cell types and correlate with HBV susceptibility

• Immunofluorescence to visualize subcellular localization during infection

• Co-immunoprecipitation to identify potential interactions between UQCR10 and viral components

This research opens possibilities for targeting UQCR10 as a novel approach to inhibit HBV infection, particularly in chronic hepatitis B cases.

How are UQCR10 antibodies utilized in investigating mitochondrial disease mechanisms?

UQCR10 antibodies provide valuable tools for studying mitochondrial disorders, particularly those involving Complex III dysfunction:

Analysis of Cytochrome b (MT-CYB) Mutations:
Research using Δ4-CYB cybrids (cells lacking functional cytochrome b) has shown that when Complex III assembly is impaired:

• CYC1 and UQCR10 remain more stable than other Complex III components (UQCRC2, UQCRFS1)

• UQCR10 distributes across multiple assembly intermediates ranging from 25 to 2,952 kDa

• UQCR10 appears to interact with components of both Complex III and Complex IV

These findings suggest UQCR10 antibodies can track Complex III assembly defects in diseases caused by MT-CYB mutations.

Supercomplex Destabilization Analysis:
Many mitochondrial diseases show altered respiratory supercomplex formation. Antibodies against UQCR10 can detect changes in:

• The ratio between free Complex III and supercomplex-incorporated Complex III

• The appearance of subcomplexes containing UQCR10

• The association between UQCR10 and other mitochondrial structures like MICOS

Diagnostic Applications:
Western blotting with UQCR10 antibodies, particularly when combined with blue native PAGE, can reveal characteristic patterns of assembly defects that may serve as diagnostic biomarkers for specific mitochondrial disorders.

What are the methodological considerations for UQCR10 antibody use in complexome profiling studies?

Complexome profiling is an advanced technique combining blue native PAGE with mass spectrometry to analyze protein complex composition. When using UQCR10 antibodies in this context, several methodological considerations are critical:

Sample Preparation:

• Detergent selection is crucial:

  • Digitonin (1-2%) best preserves supercomplexes

  • Lauryl maltoside disrupts supercomplexes but maintains individual complexes

  • Triton X-100 or SDS cause more extensive dissociation

• Protein concentration must be standardized:

  • Typically 5-10 μg protein per lane for blue native PAGE

  • Higher loading can cause migration artifacts

Gel Analysis and Protein Extraction:

• After BN-PAGE, cut the gel lane into equal slices (approximately 60-70 slices)

• Process each slice for mass spectrometry

• For western blot validation, run parallel gels under identical conditions

Data Analysis and Quantification:

• Plot protein abundance profiles across gel fractions

• Calculate the area under the peaks defined by relative peptide intensity

• Use "internal standard" experimental controls (TOM20, TOM22, citrate synthase) to normalize across samples

Research using this approach has revealed that the distribution of UQCR10 differs dramatically between normal cells and those with Complex III assembly defects. In cells with normal Complex III assembly, UQCR10 primarily migrates with assembled Complex III. In contrast, in Δ4-CYB cybrids, UQCR10 distributes across multiple peaks with molecular sizes ranging from 25 to 2,952 kDa .

What are the common problems when working with UQCR10 antibodies and how can they be resolved?

Researchers working with UQCR10 antibodies may encounter several technical challenges:

Challenge 1: Cross-reactivity with Related Proteins
Evidence suggests that some anti-UQCRQ antibodies cross-react with UQCR10 . This can lead to misinterpretation of results.

Solutions:

• Validate antibody specificity using knockout or knockdown controls

• Use epitope-tagged versions of UQCR10 to confirm band identity

• Employ multiple antibodies targeting different epitopes

• Verify results with mass spectrometry when possible

Challenge 2: Difficulty Detecting Native UQCR10 in Some Cell Lines
Research has shown post-transcriptional inhibition of UQCR10 in certain cell lines, making detection challenging .

Solutions:

• Create stable cell lines expressing UQCR10 for positive controls

• Use mitochondrial enrichment before analysis

• Consider longer exposure times for western blots

• Normalize to mitochondrial markers rather than total protein

Challenge 3: Poor Resolution of Small Molecular Weight Protein
At only 7 kDa, UQCR10 can be difficult to resolve properly on gels.

Solutions:

• Use 15-20% acrylamide gels or 4-20% gradient gels

• Choose PVDF membranes with 0.2 μm pore size rather than 0.45 μm

• Optimize transfer conditions (100V for 1 hour or 30V overnight at 4°C)

• Consider semi-dry transfer systems for efficient transfer of small proteins

Challenge 4: Inconsistent Immunoprecipitation Results
Pulling down intact complexes containing UQCR10 can be challenging.

Solutions:

• Use digitonin (1-2%) for lysis when studying intact complexes

• Increase antibody amount for small proteins (1:50 dilution recommended)

• For weak interactions, consider crosslinking before lysis

• Optimize wash conditions to balance between specificity and yield

How should researchers interpret contradictory UQCR10 data between mRNA and protein levels?

An important research finding regarding UQCR10 is the discrepancy between mRNA and protein levels in certain cell types, which creates challenges in experimental interpretation:

Documented Contradiction:
Studies have revealed that HepG2 and Huh7 cell lines show normal UQCR10 mRNA levels compared to liver tissues, but significantly reduced protein levels . This suggests post-transcriptional regulation mechanisms affecting UQCR10 expression.

Methodological Approach to Resolve This Contradiction:

• Comprehensive Expression Analysis:

  • Perform both RT-qPCR and Western blotting on the same samples

  • Include multiple positive control tissues/cells

  • Use absolute quantification methods when possible

• Investigation of Post-transcriptional Mechanisms:

  • Analyze microRNA binding sites in UQCR10 mRNA

  • Assess protein stability with cycloheximide chase experiments

  • Examine polysome association of UQCR10 mRNA

• Creation of Controlled Expression Systems:

  • Generate stable cell lines with UQCR10 expression vectors

  • Use inducible expression systems to study dose-dependent effects

  • Tag UQCR10 to distinguish endogenous vs. exogenous protein

Experimental Design to Address this Issue:
When studying UQCR10 in new cell models, researchers should first establish the relationship between mRNA and protein levels. This is particularly important when using cell lines as disease models or for mechanistic studies, as the discrepancy between mRNA and protein may lead to misinterpretation of results.

What reference data should researchers use when interpreting UQCR10 antibody results?

When interpreting results from UQCR10 antibody experiments, researchers should reference established datasets and controls:

Expected Migration Patterns in Western Blotting:

• Predicted molecular weight: 7 kDa

• Observed band in validated samples: 7 kDa

Validated Cell Lines for Positive Controls:

• HepG2 cells show clear UQCR10 expression

• HeLa, Jurkat, and Saos-2 cells also express detectable levels

Expected Distribution in Complexome Profiling:
In normal cells, UQCR10 distributes primarily with assembled Complex III, while in cells with disrupted Complex III assembly (e.g., Δ4-CYB mutants), it distributes across multiple subcomplexes of varying molecular weights .

Known Interaction Partners for Co-immunoprecipitation:
Research has identified consistent interaction partners for UQCR10:

• Strong associations: CYC1 and UQCRH (Complex III components)

• Consistent interactions: MT-CO2, COX5B, COX6C, and COX6B1 (Complex IV components)

• Context-dependent interactions: GHITM, CHCHD3 (MIC19), and HADHB

Assay-Specific Reference Values:
For blue native PAGE analysis, researchers should reference the following molecular weight ranges for UQCR10-containing complexes:

• Individual Complex III: ~500 kDa

• Complex III₂+IV: ~700 kDa

• Complex I+III₂: ~1,500 kDa

• Complex I+III₂+IV₁₋₄: 1,700-2,700 kDa

• In Complex III assembly defects: Additional subcomplexes at 25-2,952 kDa

What emerging techniques could enhance UQCR10 antibody applications in mitochondrial research?

Several cutting-edge approaches are poised to expand the utility of UQCR10 antibodies in mitochondrial research:

Advanced Imaging Approaches:

• Super-resolution microscopy combined with UQCR10 antibodies can reveal the nanoscale organization of respiratory complexes within mitochondria

• Correlative light and electron microscopy (CLEM) can connect UQCR10 localization with mitochondrial ultrastructure

• Expansion microscopy could provide enhanced resolution of UQCR10 distribution within mitochondrial subcompartments

Proximity Labeling Technologies:

• BioID or APEX2 fusion proteins with UQCR10 can map the protein's immediate neighborhood in living cells

• This approach could identify transient or weak interactions missed by traditional co-immunoprecipitation

• Time-resolved proximity labeling could capture dynamic changes in UQCR10 interactions during mitochondrial biogenesis or stress responses

Single-Cell Approaches:

• Single-cell proteomics to analyze UQCR10 levels and complex incorporation in heterogeneous populations

• Integration with spatial transcriptomics to correlate UQCR10 protein levels with local gene expression patterns

• These approaches could help understand the heterogeneity observed in UQCR10 expression within cell populations, which impacts HBV infection susceptibility

In situ Structural Studies:

• Cryogenic electron tomography (cryo-ET) combined with immunogold labeling using UQCR10 antibodies

• This could reveal the native arrangement of UQCR10 within respiratory complexes in their cellular context

• Cross-linking mass spectrometry (XL-MS) to map the structural neighborhood of UQCR10 within assembled complexes

How might UQCR10 antibodies contribute to understanding the link between mitochondrial function and viral infections?

The unexpected discovery of UQCR10's role in HBV infection opens new research directions where UQCR10 antibodies could be valuable tools:

Mechanistic Studies:

• Co-immunoprecipitation with UQCR10 antibodies followed by viral protein detection could identify direct interactions between UQCR10 and viral components

• Time-course studies with UQCR10 antibodies could track changes in localization or complex incorporation during viral infection

• Comparative studies across different viruses could determine if UQCR10's role in infection is HBV-specific or more general

Therapeutic Target Validation:

• UQCR10 antibodies could help evaluate whether interventions targeting this protein affect viral replication without disrupting mitochondrial function

• Epitope mapping with different UQCR10 antibodies might identify specific regions involved in viral interactions versus mitochondrial functions

Connection to Immune Signaling:

• Mitochondria are increasingly recognized as hubs for innate immune signaling

• UQCR10 antibodies could help investigate whether its role in HBV infection connects to mitochondrial antiviral signaling pathways

• Co-localization studies with immune signaling components during infection could reveal functional interactions

Clinical Translation:

• UQCR10 antibodies could be used to assess protein levels in patient-derived samples

• This might help stratify patients based on potential susceptibility to HBV infection or response to therapies

• Serial monitoring of UQCR10 in infected versus treated states could provide biomarkers for treatment response

The research finding that transgenic HBV preferentially replicates in cells with higher UQCR10 levels suggests a direct functional relationship . UQCR10 antibodies will be essential tools in unraveling this unexpected connection between mitochondrial function and viral infection.