UQCRB Antibody

What is UQCRB and why is it important in cellular research?

UQCRB (Ubiquinol-cytochrome c reductase binding protein) is a 13.3-kDa subunit of mitochondrial complex III, located at human chromosome 8q22. It plays critical roles in:

• Mitochondrial complex III stability

• Electron transport chain functionality

• Cellular oxygen sensing mechanisms

• Angiogenesis regulation

UQCRB is found in respiratory chains of all aerobic organisms and participates in stabilizing the ubisemiquinone radical through hydrophobic interactions with ubiquinone. Deletion of the gene encoding UQCRB causes defects in complex III function, resulting in hypoglycemia and lactic acidosis, highlighting its physiological significance .

Which applications are UQCRB antibodies commonly used for?

UQCRB antibodies are validated for multiple research applications:

The optimal dilution should be determined for each specific antibody and experimental condition .

What species reactivity is available for UQCRB antibodies?

Commercial UQCRB antibodies are available with reactivity against:

• Human

• Mouse

• Rat

Some antibodies also have predicted reactivity with additional species due to sequence homology, including:

• Pig

• Zebrafish

• Bovine

• Horse

• Sheep

• Rabbit

• Dog

• Chicken

• Xenopus

Always verify actual reactivity through validation experiments for your specific species of interest .

How should I optimize immunohistochemical detection of UQCRB in tissue samples?

For optimal IHC detection of UQCRB:

• Tissue preparation: Use 4-μm thick sections from paraffin-embedded blocks mounted on coated slides.

• Deparaffinization and rehydration: Process slides through xylene and graded alcohol washes.

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: 10 mM citrate buffer (pH 6.0) in a pressure boiler for 10 minutes

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide.

• Primary antibody incubation: Apply rabbit polyclonal UQCRB antibody (typically 1:200 dilution) overnight at 4°C.

• Secondary antibody and detection:

  • Incubate with appropriate anti-rabbit secondary antibody for 30 minutes

  • Use horseradish peroxidase complex method for visualization

  • Develop with DAB (3,3'-Diaminobenzidine) substrate

• Counterstaining: Hematoxylin is recommended for nuclear counterstaining .

What are the best practices for Western blotting detection of UQCRB?

For optimal Western blotting results with UQCRB:

• Sample preparation:

  • For tissue samples: Heart tissue from mouse or rat provides strong signal

  • For cells: Mitochondria-rich cell lines yield better results

• Protein loading: UQCRB is a low molecular weight protein (14 kDa); use appropriate percentage gels (12-15%)

• Antibody selection and dilution:

  • Monoclonal antibodies: 0.1-1.0 μg/ml

  • Polyclonal antibodies: 1:500-1:2000 dilution

• Molecular weight verification: UQCRB should appear at approximately 14 kDa

• Controls:

  • Positive control: Heart tissue lysate

  • Loading control: Other mitochondrial proteins or housekeeping genes (e.g., GAPDH)

• Detection system: Enhanced chemiluminescence (ECL) is suitable; use 5 μL of quality control per well .

How can I use UQCRB antibodies for studying protein-protein interactions within mitochondrial complex III?

For studying UQCRB interactions within complex III:

• Co-immunoprecipitation (Co-IP):

  • Use 0.5-4.0 μg of UQCRB antibody per 1-3 mg of total protein lysate

  • Mild lysis buffers containing 0.5-1% NP-40 or Triton X-100 are recommended to preserve protein complexes

  • Include protease inhibitors to prevent degradation

  • Verify interactions with reciprocal Co-IP when possible

• Proximity ligation assay (PLA):

  • Useful for detecting protein-protein interactions in situ

  • Requires antibodies from different species for UQCRB and its potential interaction partners

• Blue native electrophoresis:

  • Enables analysis of intact mitochondrial complexes

  • Can be followed by second-dimension SDS-PAGE for subunit analysis

• Cross-linking studies:

  • Chemical cross-linkers can stabilize transient interactions

  • Follow with immunoprecipitation using UQCRB antibodies .

How can UQCRB antibodies be used to investigate its role as a prognostic biomarker in colorectal cancer?

For investigating UQCRB as a colorectal cancer biomarker:

• Tissue microarray (TMA) analysis:

  • Compare UQCRB expression between CRC tissues and matched adjacent non-tumor tissues

  • Correlate expression with clinical staging and patient outcomes

  • Use IHC scoring systems (e.g., H-score or Allred score) for quantification

• Gene expression correlation:

  • Measure copy number variation (CNV) of UQCRB (reported 1.32-fold increase in CRC tissues)

  • Analyze gene expression in relationship to protein levels

  • Examine correlations with other complex III genes (UQCRFS1, MT-CYB)

• Methylation analysis:

  • Examine DNA methylation of the UQCRB promoter (often hypermethylated in CRC)

  • Correlate with expression and clinical outcomes

• SNP association:

  • Investigate polymorphisms in UQCRB (particularly rs7836698 in the 3'-UTR region)

  • Analyze association with CRC clinical stage

• ROC curve analysis:

  • Evaluate sensitivity and specificity (reported as 93.3% and 100.0% respectively)

  • Calculate AUC values (reported as 0.991 for UQCRB expression) .

What methods can be used to study UQCRB's role in angiogenesis using small molecule inhibitors?

To investigate UQCRB's role in angiogenesis using small molecule inhibitors:

• Small molecule inhibitor approach:

  • Terpestacin has been identified as a UQCRB-targeting small molecule

  • Use both genetic (RNA interference) and pharmacological approaches to compare effects

• Hypoxia-induced ROS measurement:

  • Monitor reactive oxygen species levels using fluorescent probes

  • Compare effects of UQCRB inhibition or silencing on ROS production

• Angiogenesis assays:

  • Tube formation assay with endothelial cells

  • Chick chorioallantoic membrane (CAM) assay

  • Matrigel plug assay in mice

• VEGF signaling analysis:

  • UQCRB regulates VEGFR2 signaling in endothelial cells

  • Measure VEGF expression and VEGFR2 phosphorylation

• Oxygen consumption rate (OCR):

  • Measure mitochondrial respiration using Seahorse technology

  • Compare effects of genetic silencing versus chemical inhibition .

How can I use circulating miRNAs as biomarkers in relation to UQCRB expression in cancer?

For studying circulating miRNAs as biomarkers related to UQCRB:

• miRNA identification:

  • miR-4435 has been identified as a UQCRB-related circulating miRNA

  • Use qRT-PCR to quantify miRNA levels in patient samples

• Exosome isolation and analysis:

  • Isolate exosomes from cell culture medium (CCM) of UQCRB-expressing cells

  • Extract exosomes from human serum samples using ultracentrifugation or commercial kits

  • Analyze miRNA content using qRT-PCR

• miRNA inhibitor transfection:

  • Transfect cells using Lipofectamine RNAiMAX with miR-4435 inhibitor

  • Measure effects on cell proliferation using MTT assay

• Migration assay:

  • Use wound healing assay to evaluate cell migration ability

  • Create artificial wounds by scratching cell monolayers with pipette tips

  • Measure migration distance using ImageJ software

• Target validation:

  • Identify miRNA targets using mRNA-sequencing and miRDB analysis

  • Tumor suppressor gene TIMP3 has been identified as a target of miR-4435 .

How can I resolve specificity issues when using UQCRB antibodies in complex tissue samples?

To improve specificity when using UQCRB antibodies:

• Antibody validation:

  • Verify antibody specificity using knockout/knockdown controls

  • Perform peptide competition assays to confirm epitope specificity

  • Cross-validate results with multiple antibodies targeting different epitopes

• Optimization strategies:

  • Titrate antibody concentration (start with manufacturer's recommended range)

  • Optimize blocking conditions (3-5% BSA or normal serum from the same species as secondary antibody)

  • Include additional washing steps to reduce background

• Sample preparation improvements:

  • For mitochondrial proteins, consider isolating mitochondrial fractions

  • Use fresh samples when possible (avoid multiple freeze-thaw cycles)

  • Include protease inhibitors during extraction

• Detection system considerations:

  • For IHC, compare DAB vs. fluorescence-based detection

  • For low abundance detection, consider signal amplification methods

  • Use secondary antibodies with minimal cross-reactivity .

What controls should I include when studying UQCRB knockdown effects on mitochondrial function?

Essential controls for UQCRB knockdown studies:

• Knockdown validation controls:

  • Verify knockdown efficiency at both mRNA (qRT-PCR) and protein levels (Western blot)

  • Include scrambled/non-targeting RNA controls

  • Use multiple shRNA/siRNA sequences to rule out off-target effects

• Functional controls:

  • Empty vector controls for viral transduction

  • Rescue experiments with UQCRB overexpression to verify phenotype specificity

  • Time-course analysis to distinguish primary from secondary effects

• Mitochondrial function controls:

  • Measure effects on other complex III components (UQCRFS1, UQCRC2)

  • Include mitochondrial mass markers (VDAC, TOM20) to normalize for mitochondrial content

  • Compare with chemical inhibitors of complex III (antimycin A, myxothiazol)

• Cell viability monitoring:

  • Monitor cell death/apoptosis markers

  • Check for compensatory mechanisms that might be activated .

How do post-translational modifications affect UQCRB detection, and how can I account for them?

Post-translational modifications (PTMs) affecting UQCRB detection:

• Known UQCRB PTMs:

  • Acetylation (K4)

  • Ubiquitination (K4)

  • Phosphorylation (S8)

• Detection strategies:

  • Use modification-specific antibodies when available

  • For phosphorylation studies, include phosphatase inhibitors during sample preparation

  • For ubiquitination studies, add proteasome inhibitors (MG132) before cell lysis

• Experimental approaches:

  • Phosphatase treatment of samples can confirm phosphorylation-dependent band shifts

  • Immunoprecipitation followed by Western blotting with PTM-specific antibodies

  • Mass spectrometry analysis for comprehensive PTM profiling

• Interpreting multiple bands:

  • Multiple bands may represent different PTM states

  • Compare band patterns in different cell types or experimental conditions

  • Use site-directed mutagenesis (K4R, S8A) to confirm PTM sites .

How can UQCRB antibodies be used to investigate mitochondrial dysfunction in cancer metabolism?

For studying UQCRB in cancer metabolism:

• Metabolic profiling approaches:

  • Compare OXPHOS dependency using Seahorse analysis in UQCRB-high vs. UQCRB-low cancer cells

  • Measure lactate production to assess glycolytic shift

  • Analyze TCA cycle intermediates using mass spectrometry

• ROS production assessment:

  • Measure mitochondrial ROS using MitoSOX Red

  • Compare basal vs. stress-induced ROS levels

  • Correlate with UQCRB expression or inhibition

• Hypoxia adaptation studies:

  • Compare UQCRB expression in normoxic vs. hypoxic conditions

  • Evaluate HIF-1α stabilization in relation to UQCRB levels

  • Assess hypoxia-induced EMT markers (Snail, E-cadherin)

• Therapeutic targeting strategies:

  • Combine UQCRB inhibition with glycolysis inhibitors

  • Test sensitization to conventional chemotherapeutics

  • Evaluate anti-angiogenic effects through endothelial cell co-culture models .

What is the relationship between UQCRB and stem cell properties in cancer, and how can it be studied?

To investigate UQCRB's role in cancer stem cell properties:

• Cancer stem cell (CSC) isolation:

  • Use flow cytometry to isolate CD44+/CD24- breast cancer stem cells

  • Isolate side population cells using Hoechst 33342 dye exclusion

  • Analyze UQCRB expression in CSC vs. non-CSC populations

• Sphere formation assays:

  • Compare tumorsphere formation ability after UQCRB knockdown

  • Assess serial passage capability to evaluate self-renewal

• Stem cell marker analysis:

  • Measure expression of stemness markers (SOX2, OCT4, NANOG)

  • Correlate with UQCRB expression levels

  • Use immunofluorescence co-staining with UQCRB and stem cell markers

• In vivo limiting dilution assays:

  • Inject decreasing numbers of cells with UQCRB knockdown vs. controls

  • Calculate tumor-initiating cell frequency

• Mitochondrial dynamics:

  • Analyze mitochondrial morphology in CSCs vs. non-CSCs

  • Examine correlation between UQCRB expression and mitochondrial fusion/fission proteins .

How does UQCRB contribute to cellular senescence in stem cells, and what experimental approaches can investigate this relationship?

To study UQCRB's role in stem cell senescence:

• Senescence marker analysis:

  • SA-β-galactosidase staining following UQCRB knockdown

  • p16INK4a and p21 expression measurement

  • Colony formation assays to assess proliferative capacity

• CRISPR/Cas9-mediated gene editing:

  • Generate UQCRB knockout in human mesenchymal stem cells (hMSCs)

  • Compare with UQCRC2 knockout effects

  • Rescue experiments with UQCRB overexpression

• Mitochondrial function assessment:

  • Measure mitochondrial ROS accumulation using fluorescent probes

  • Analyze mitochondrial membrane potential

  • Assess electron transport chain complex activities

• Molecular pathway analysis:

  • Investigate EIF4EBP1 (4E-BP1) relationship with UQCRB

  • Examine downstream effects on other complex III components (UQCRC2, UQCRFS1)

  • Analyze translation regulation of mitochondrial proteins

• Therapeutic intervention studies:

  • Test antioxidant effects on UQCRB-deficiency induced senescence

  • Evaluate mTOR inhibitors in the context of UQCRB expression

  • Examine mitochondrially-targeted interventions .