uqcc3 Antibody

What is UQCC3 and why is it significant in biomedical research?

UQCC3 (Ubiquinol-Cytochrome C Reductase Complex Assembly Factor 3, also known as C11orf83) is a small (10.1 kDa, 93 amino acids) mitochondrial protein that functions in the assembly and stability of mitochondrial respiratory chain supercomplexes, particularly complex III. Research has revealed UQCC3's critical importance in:

• Embryonic development and angiogenesis, with knockout studies showing embryonic lethality in mice at 9.5-10.5 days postcoitum due to dramatically reduced vessel density

• Tumor growth and angiogenesis through regulation of the ROS/HIF/VEGF pathway

• Cellular adaptation to hypoxia, being upregulated under hypoxic conditions

• Mitochondrial homeostasis and reactive oxygen species (ROS) generation

UQCC3 has emerged as a promising research target due to its implications in cancer progression, with higher expression levels associated with poor prognosis in multiple tumor types . Additionally, recent research has shown that UQCC3 can translocate to the nucleus under hypoxic conditions, suggesting regulatory functions beyond its mitochondrial role .

What is known about UQCC3's structure and localization?

UQCC3's structural and localization characteristics include:

• Gene location: Chromosome 11q12.3, spanning 2,036 base pairs

• Protein composition: 93 amino acids with a molecular weight of approximately 10.1 kDa

• Structural elements:

  • N-terminal signal peptide

  • Signal transmembrane structure

  • Multiple phosphorylation sites

  • Secondary structure composed mostly of random coils and alpha helices

  • Alpha helices 2 and 3 bind to cardiolipin

Subcellular localization:

• Under normoxic conditions: Predominantly mitochondrial, facing the intermembrane space

• Under hypoxic conditions: Both mitochondrial and nuclear localization

• Nuclear translocation mechanism: Contains a bipartite nuclear localization signal (NLS) at amino acids 30-61 that becomes accessible after cleavage by PARL protease under hypoxia

What applications are UQCC3 antibodies used for in research?

UQCC3 antibodies are utilized in several research applications:

• Western Blot Analysis:

  • Detection of full-length UQCC3 (~10.1 kDa)

  • Identification of cleaved forms under hypoxia (<35 kDa)

  • Comparative analysis of expression levels between normal and pathological samples

  • Typical dilutions: 0.04-0.4 μg/mL

• Immunohistochemistry (IHC):

  • Visualization of tissue distribution patterns

  • Assessment of subcellular localization

  • Evaluation of expression in tumor vs. normal tissues

  • Recommended dilutions: 1:500-1:1000

• Immunofluorescence:

  • High-resolution imaging of subcellular localization

  • Co-localization studies with mitochondrial or nuclear markers

  • Live-cell imaging using UQCC3-EGFP fusion proteins

• ELISA:

  • Quantitative measurement of UQCC3 levels in biological samples

  • Test range: 0.313-20 ng/mL (typical for commercial kits)

• Research Applications:

  • Study of angiogenesis in development and cancer progression

  • Investigation of hypoxic response mechanisms

  • Analysis of mitochondrial complex III assembly and function

  • Evaluation as a potential prognostic biomarker in cancers

What are the recommended protocols for Western blot detection of UQCC3?

Sample preparation considerations:

• For total protein: Use RIPA or NP-40 based lysis buffers with protease inhibitors

• For mitochondrial enrichment: Employ specific mitochondrial isolation protocols

• For nuclear UQCC3 detection: Perform subcellular fractionation to isolate nuclear proteins

Optimized Western blot protocol:

• Sample preparation:

  • Load 20-50 μg protein in reducing sample buffer

  • Include both normoxic and hypoxic samples for comparison

• Electrophoresis:

  • Use 12-15% acrylamide gels for optimal separation of small proteins (~10.1 kDa)

  • For detecting both full-length and cleaved forms, use high-resolution gels

• Transfer:

  • Transfer to PVDF membrane (recommended over nitrocellulose for small proteins)

  • Use low methanol transfer buffer (5-10%) to improve transfer of small proteins

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary UQCC3 antibody at manufacturer's recommended dilution (typically 0.04-0.4 μg/mL ) overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody

• Detection:

  • Use high-sensitivity ECL reagents for optimal detection of low-abundance proteins

  • For cleaved forms detection, longer exposure times may be needed

Expected results:

• Full-length UQCC3: ~10.1 kDa band

• Cleaved form under hypoxia: <35 kDa band

• UQCC3-EGFP fusion proteins: ~37 kDa

How can researchers optimize immunohistochemistry for UQCC3 detection?

Tissue processing and antigen retrieval:

• Fix tissues with 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard protocols

• Section at 4-5 μm thickness

• Perform heat-induced epitope retrieval:

  • Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

  • Optimize heating time (typically 15-20 minutes)

Staining protocol optimization:

• Deparaffinize and rehydrate sections

• Block endogenous peroxidase with 3% H₂O₂

• Perform antigen retrieval as determined optimal

• Block non-specific binding:

  • 5-10% normal serum from secondary antibody species

  • Consider adding 0.1-0.3% Triton X-100 for improved antibody penetration

• Primary antibody incubation:

  • Test dilution range (1:500-1:1000 recommended for many UQCC3 antibodies )

  • Optimize incubation time and temperature

• Detection system:

  • Polymer-based systems generally provide better signal-to-noise ratio than ABC methods

  • For dual localization studies, consider fluorescent secondary antibodies

Special considerations for UQCC3:

• For detecting nuclear UQCC3, ensure proper antigen retrieval and membrane permeabilization

• When studying hypoxia-induced translocation, compare normoxic vs. hypoxic tissues

• For tumor studies, analyze both hypoxic and normoxic regions within the tumor (can be identified with hypoxia markers)

How can researchers detect both mitochondrial and nuclear UQCC3 simultaneously?

Detection of UQCC3's dual localization requires specialized techniques:

Immunofluorescence approach:

• Cell/tissue preparation:

  • For cultured cells: Grow on coverslips, induce hypoxia if studying translocation

  • For tissues: Prepare frozen sections or perform antigen retrieval on FFPE sections

• Fixation and permeabilization:

  • 4% paraformaldehyde (10-15 minutes) followed by 0.1-0.3% Triton X-100

  • Alternative: Methanol fixation (-20°C, 10 minutes) for simultaneous fixation and permeabilization

• Staining:

  • UQCC3 antibody (optimal dilution determined empirically)

  • Mitochondrial marker (e.g., TOMM20, COX IV, MitoTracker)

  • Nuclear counterstain (e.g., DAPI, Hoechst)

• Imaging:

  • Confocal microscopy with Z-stacking for 3D localization

  • High-resolution imaging to distinguish mitochondrial and nuclear signals

  • Quantitative fluorescence analysis to measure relative intensities in each compartment

Subcellular fractionation approach:

• Prepare purified mitochondrial, cytosolic, and nuclear fractions

• Perform Western blot analysis of each fraction

• Probe for UQCC3 along with compartment-specific markers:

  • Mitochondrial markers: VDAC, COX IV

  • Nuclear markers: Lamin B, Histone H3

  • Cytosolic markers: GAPDH, β-tubulin

• Compare band patterns and intensities across fractions and conditions

UQCC3-GFP fusion protein approach:

• Generate constructs for full-length UQCC3 (1-93) and NLS-containing fragments (30-61 or 30-93)

• Transfect cells and observe localization under normoxia and hypoxia

• Perform live-cell imaging to track dynamic changes during hypoxia induction

What controls should be included when studying UQCC3?

Rigorous controls are essential for reliable UQCC3 research:

Genetic controls:

• Negative controls:

  • UQCC3 knockout cells/tissues (TALEN or CRISPR/Cas9-generated)

  • siRNA/shRNA knockdown samples (significant reduction shown in search results)

  • No primary antibody control for IHC/IF

• Positive controls:

  • Tissues known to express UQCC3 (testis shows moderate cytoplasmic granular positivity)

  • Overexpression systems (cells transfected with UQCC3 expression vectors)

  • Recombinant UQCC3 protein

Experimental condition controls:

• Hypoxia studies:

  • Paired normoxic and hypoxic samples

  • Time course samples (4h, 8h, etc.) to track expression changes

  • Hypoxia marker verification (e.g., HIF-1α stabilization)

• Specificity controls:

  • Peptide competition assay (pre-incubating antibody with immunizing peptide)

  • Multiple antibodies targeting different UQCC3 epitopes

  • Isotype controls at matching concentrations

Subcellular localization controls:

• Compartment markers:

  • Mitochondrial markers (e.g., TOMM20, COX IV)

  • Nuclear markers (e.g., Lamin B, DAPI)

  • Purity control for fractionation (compartment-specific markers)

• Translocation controls:

  • PARL knockdown to prevent cleavage and nuclear translocation

  • UQCC3 mutants with altered NLS sequence

  • MitoQ treatment to assess ROS-dependent effects

How does UQCC3 regulate the ROS/HIF/VEGF pathway in angiogenesis?

UQCC3 plays a crucial role in angiogenesis through a complex molecular pathway:

Mitochondrial function and ROS generation:

• UQCC3 is involved in complex III assembly and stability

• Knockout of UQCC3 reduces mitochondrial ROS production, as detected by MitoSOX

• UQCC3 knockout also decreases:

  • Cellular ATP content

  • Mitochondrial mass

  • Mitochondrial membrane potential

ROS-dependent HIF-1α stabilization:

• Under hypoxia, UQCC3 levels increase (slightly after 4h, significantly after 8h)

• UQCC3-generated ROS is required for HIF-1α stabilization

• UQCC3 knockout leads to decreased HIF-1α intensity in both embryos and tumor cells

• MitoQ (a mitochondrial ROS scavenger) prevents UQCC3-mediated increases in VEGF

VEGF expression and angiogenesis:

• HIF-1α activates VEGF transcription

• UQCC3-deleted embryos show dramatically reduced VEGF expression

• In tumor cells, UQCC3 knockout results in less VEGF secretion

• The angiogenic effect is VEGF-dependent, as shown in tube formation assays:

  • HUVEC tube formation is impaired in UQCC3-knockout tumor cell medium

  • This impairment can be rescued by VEGF supplementation

Embryonic and tumor angiogenesis:

• In mouse embryos, UQCC3 deletion causes:

  • Absence of large, branched blood vessels in yolk sacs

  • Reduction of vessel branch points

  • Increased apoptosis

• In zebrafish, uqcc3 knockout impairs vascular development

• In tumors, UQCC3 knockout results in:

  • Slower tumor growth

  • Reduced vessel density (as shown by CD31 and Dextran-FITC staining)

Mechanistic pathway diagram:
UQCC3 upregulation in hypoxia → Enhanced mitochondrial ROS generation → HIF-1α stabilization → Increased VEGF expression → Angiogenesis → Tumor growth/embryonic development

What is the significance of UQCC3 nuclear translocation under hypoxia?

Recent research has uncovered a novel function of UQCC3 involving its translocation to the nucleus under hypoxic conditions:

Mechanism of nuclear translocation:

• UQCC3 contains a bipartite nuclear localization signal (NLS) at amino acids 30-61

• Under hypoxia, UQCC3 is cleaved in mitochondria by PARL protease, revealing its NLS

• The cleaved form (<35 kDa vs. full-length 37 kDa) translocates to the nucleus

• Silencing PARL eliminates the cleaved band and prevents nuclear translocation

• Alanine scanning mutagenesis identified amino acids 26-29 as crucial for normal cleavage

Functional consequences of nuclear translocation:

• Enhanced cellular proliferation:

  • Cells expressing nuclear-targeted UQCC3 (30-93) show growth advantage over full-length UQCC3 (1-93)

  • This growth advantage occurs under both normoxia and hypoxia

• Accelerated tumor growth:

  • Xenografts expressing nuclear-targeted UQCC3 reach significantly larger volumes

  • By day 20, UQCC3(30-93) xenografts: 1501.15 mm³

  • By day 20, UQCC3(1-93) xenografts: 987.90 mm³

• Clinical correlation with poor prognosis:

  • 33.3% of HCC tumor samples show positive nuclear UQCC3 staining

  • Median survival with nuclear-positive UQCC3: 18 months

  • Median survival with nuclear-negative UQCC3: 43 months

  • Significant difference in survival curves (p=0.0240)

Research implications:

• Represents a novel mitochondrial retrograde signaling pathway

• Highlights UQCC3 as a nuclear factor affecting tumor adaptation to hypoxia

• Suggests nuclear UQCC3 may serve as a prognostic biomarker

• Identifies a potential new therapeutic target for liver cancer

How can researchers effectively study UQCC3's role in complex III assembly?

Investigating UQCC3's function in mitochondrial complex III assembly requires specialized techniques:

Genetic manipulation approaches:

• Knockout models:

  • TALEN-mediated knockout in mice

  • CRISPR/Cas9-mediated knockout in zebrafish

  • Liver-specific conditional (Cre-loxp system) knockout in mice

  • CRISPR/Cas9 or siRNA knockdown in cell lines

• Expression systems:

  • Tetracycline-inducible expression of UQCC3-FLAG

  • UQCC3-EGFP fusion proteins (full-length and truncated variants)

Biochemical and functional assays:

• Complex III assembly:

  • Blue Native PAGE to analyze intact complexes and supercomplexes

  • Immunoprecipitation to study interaction with core complex III subunits

  • Analysis of UQCC3 relationship with other assembly factors (UQCC1, UQCC2)

  • Assessment of complex III levels after UQCC3 manipulation

• Mitochondrial function:

  • Mitochondrial respiration measurements

  • ATP production assays

  • Mitochondrial membrane potential analysis

  • ROS production (MitoSOX staining)

  • Mitochondrial mass quantification

Protein-protein interaction studies:

• Co-immunoprecipitation:

  • Using UQCC3 antibodies to pull down interacting proteins

  • Using antibodies against complex III components to detect UQCC3 interaction

  • FLAG-tagged UQCC3 for affinity purification

• Proximity labeling:

  • APEX2 or BioID fusion with UQCC3 to identify proximal proteins

  • Focused on mitochondrial intermembrane space interactions

• Structural studies:

  • Cryo-EM of complex III with and without UQCC3

  • Analysis of UQCC3 binding to cardiolipin via alpha helices 2 and 3

Experimental design considerations:

• Study UQCC3 in relationship to other assembly factors (UQCC1, UQCC2)

• Investigate temporal sequence of complex III assembly

• Analyze effects on other respiratory complexes and supercomplexes

• Consider tissue-specific differences in complex III assembly

Why might Western blots show multiple or unexpected UQCC3 bands?

Multiple or unexpected bands in UQCC3 Western blots can result from several factors:

Biological causes:

• Proteolytic processing:

  • Full-length UQCC3: ~10.1 kDa

  • Cleaved fragment under hypoxia: <35 kDa

  • UQCC3-EGFP fusion: ~37 kDa (full-length)

  • The cleaved form increases under hypoxia and is present in nuclear fractions

• Post-translational modifications:

  • Phosphorylation at multiple sites may cause mobility shifts

  • Other modifications may occur under specific cellular conditions

• Different protein variants:

  • Nuclear vs. mitochondrial forms

  • Potential alternative splicing variants (though not specifically mentioned in the research)

Technical causes:

• Sample preparation issues:

  • Protein degradation during extraction

  • Insufficient denaturation

  • Incomplete reduction of disulfide bonds

• Antibody specificity:

  • Cross-reactivity with similar proteins

  • Recognition of non-specific epitopes

  • Binding to both native and denatured forms

Troubleshooting strategies:

• Compare experimental conditions:

  • Normoxia vs. hypoxia samples (cleaved form increases in hypoxia)

  • Subcellular fractions (mitochondrial vs. nuclear)

  • UQCC3 knockout vs. wild-type samples

• Optimize sample preparation:

  • Use fresh samples with complete protease inhibitor cocktails

  • Ensure thorough denaturation (adequate SDS, heating)

  • Consider phosphatase treatment to eliminate phosphorylation-based shifts

• Validate antibody specificity:

  • Test multiple antibodies targeting different epitopes

  • Perform peptide competition assay

  • Include known positive and negative controls

• Gel optimization:

  • Use gradient gels for better separation of multiple forms

  • Adjust running conditions for small proteins (~10 kDa)

  • Consider specialized gel systems for low molecular weight proteins

How can researchers troubleshoot poor or inconsistent UQCC3 immunostaining?

Challenges with UQCC3 immunostaining can be addressed through systematic troubleshooting:

Common issues and solutions:

• Weak or absent signal:

  • Antigen retrieval: Test multiple methods (citrate pH 6.0 vs. EDTA pH 9.0)

  • Antibody concentration: Try increasing concentration or longer incubation

  • Detection system: Switch to more sensitive detection method

  • Sample processing: Ensure fixation isn't excessive (overfixation can mask epitopes)

  • Antibody validation: Confirm antibody works in your specific application/species

• High background:

  • Blocking: Increase blocking time/concentration or try different blocking agents

  • Antibody dilution: Use more diluted antibody solution

  • Washing: Increase number/duration of wash steps

  • Endogenous enzymes: Block endogenous peroxidase more thoroughly

  • Secondary antibody: Test alternative secondary antibody or detection system

• Non-specific staining:

  • Antibody specificity: Validate with UQCC3 knockout controls

  • Cross-reactivity: Use IgG isotype control at same concentration

  • Tissue processing: Optimize fixation time and conditions

  • Antigen retrieval: Adjust time and conditions to minimize epitope alteration

• Inconsistent results:

  • Standardize protocol: Document and strictly follow optimized protocol

  • Tissue quality: Ensure consistent tissue collection and processing

  • Controls: Include positive and negative controls in every experiment

  • Antibody storage: Aliquot antibodies to avoid freeze-thaw cycles

  • Batch testing: Process all comparative samples in same batch

Optimization strategy for UQCC3 immunostaining:

• Start with manufacturer's recommended protocol

• Systematically test variables one at a time:

  • Antigen retrieval method and time

  • Antibody dilution series (1:250, 1:500, 1:1000, 1:2000)

  • Incubation time and temperature

  • Detection system alternatives

• Once optimized, document detailed protocol and standardize for all experiments

• Always include appropriate controls:

  • Known positive tissue (e.g., testis shows moderate cytoplasmic positivity)

  • Negative control (UQCC3 knockout or no primary antibody)

  • Subcellular localization controls (for nuclear vs. mitochondrial staining)

What specialized techniques can help detect low abundance or translocating UQCC3?

Detecting low abundance UQCC3 or its translocation between compartments requires advanced techniques:

Signal amplification methods:

• Tyramide signal amplification (TSA):

  • Enhances sensitivity 10-200 fold over conventional methods

  • Particularly useful for detecting low abundance proteins

  • Compatible with multiple detection systems

• Proximity ligation assay (PLA):

  • Provides single-molecule detection sensitivity

  • Useful for confirming UQCC3 interactions with other proteins

  • Can detect proteins in specific subcellular contexts

• Highly sensitive detection reagents:

  • Super-sensitive ECL substrates for Western blot

  • Quantum dot-conjugated antibodies for fluorescence

  • Polymer-based detection systems for IHC

Enrichment strategies:

• Subcellular fractionation:

  • Separate mitochondrial, cytoplasmic, and nuclear fractions

  • Concentrate UQCC3 from each compartment

  • Use of high-purity isolation kits for cleaner fractions

• Immunoprecipitation:

  • Enrich UQCC3 from lysates before analysis

  • Can be combined with mass spectrometry for interaction studies

  • Useful for detecting modified forms of UQCC3

Advanced imaging approaches:

• Super-resolution microscopy:

  • STORM, PALM, or STED microscopy for nanoscale localization

  • Allows visualization of UQCC3 within mitochondrial subcompartments

  • Can resolve translocation events at high resolution

• Live-cell imaging:

  • UQCC3-fluorescent protein fusions to track dynamics

  • Photoactivatable or photoconvertible tags to follow specific populations

  • Time-lapse imaging during hypoxia induction to capture translocation

• FRET/FLIM techniques:

  • Detect UQCC3 proximity to other proteins in living cells

  • Monitor conformational changes during translocation

  • Quantify protein-protein interactions in different cellular compartments

Special considerations for UQCC3 translocation:

• Dual labeling strategies:

  • Antibodies recognizing different domains (N-terminal vs. C-terminal)

  • Differential detection of full-length vs. cleaved forms

  • Correlation with PARL protease activity/localization

• Time-course analysis:

  • Capture transitional states during hypoxia response

  • Document progressive changes in localization

  • Correlate with ROS levels and HIF-1α stabilization

Future directions in UQCC3 research

Research on UQCC3 is expanding into several promising directions:

• Therapeutic targeting:

  • Development of small molecule inhibitors of UQCC3

  • Investigation of PARL inhibitors to prevent nuclear translocation

  • Exploration of UQCC3 as a biomarker for cancer prognosis and treatment response

• Molecular mechanisms:

  • Identification of nuclear UQCC3 target genes and functions

  • Further characterization of the mitochondria-to-nucleus retrograde signaling pathway

  • Understanding of tissue-specific roles in development and disease

• Methodological advances:

  • Development of more specific antibodies for various UQCC3 domains and forms

  • Creation of conditional and inducible knockout models for temporal studies

  • Application of spatial transcriptomics to analyze UQCC3-dependent gene expression

• Clinical applications:

  • Evaluation of nuclear UQCC3 as a prognostic marker across cancer types

  • Investigation of UQCC3 in other hypoxia-related pathologies beyond cancer

  • Exploration of genetic UQCC3 variants in mitochondrial and developmental disorders