COX4I2 Antibody

What is COX4I2 and why is it significant in research?

COX4I2 (Cytochrome c oxidase subunit 4 isoform 2) is a component of cytochrome c oxidase, which functions as the terminal enzyme in the mitochondrial electron transport chain driving oxidative phosphorylation. It catalyzes the transfer of electrons from reduced cytochrome c to oxygen . The protein is particularly significant because:

• It is primarily expressed in tissues with high oxygen demand such as heart and skeletal muscles

• The COX4 subunit optimizes respiratory chain function through differential expression of its isoforms COX4I1 and COX4I2

• It plays a critical role in oxygen sensing and hypoxic response mechanisms

• Research has identified it as a biomarker for blood supply in adrenal tumors

What applications are validated for COX4I2 antibodies?

COX4I2 antibodies have been validated for multiple experimental applications with specific positive detections in various cell lines and tissues:

Sources:

What is the difference between COX4I1 and COX4I2 isoforms?

The two isoforms differ in several key aspects:

• Oxygen affinity: COX4I2-containing enzyme shows decreased oxygen affinity compared to COX4I1, with a twofold increase in p50 (partial pressure of oxygen at half-maximal respiration)

• Tissue expression patterns: COX4I1 is ubiquitously expressed, while COX4I2 shows tissue-specific expression predominantly in tissues with high oxygen demand

• Function under hypoxia: COX4I2 expression increases under hypoxic conditions, enhancing enzyme activity while decreasing ROS production from mitochondria

• Molecular structure: COX4I2 contains unique cysteine residues at positions 40, 54, and 108 that are not present in COX4I1, which may contribute to functional differences

How should I design experiments to study COX4I2 expression in tissue samples?

For optimal results when investigating COX4I2 expression in tissue samples:

• Antibody selection: Choose antibodies validated for your specific application (WB, IHC, IF) and tissue type. Polyclonal antibodies like 11463-1-AP show reactivity with human, mouse, and rat samples

• Tissue preparation for IHC:

  • Use antigen retrieval with TE buffer pH 9.0 as primarily recommended

  • Alternative: antigen retrieval with citrate buffer pH 6.0

  • Dilution range: 1:20-1:200 for immunohistochemistry applications

• Controls:

  • Positive controls: Include HepG2 cells, human brain tissue, or human heart tissue which show consistent expression

  • Negative controls: Consider COX4I2 knockout or knockdown samples to confirm specificity

• Quantification methods:

  • For mRNA analysis: Use quantitative real-time PCR (qPCR) as performed in adrenal tumor studies

  • For protein expression: Combine Western blot quantification with immunohistochemical scoring systems (intensity grading from 0-3 as used in adrenal tumor research)

What methodological considerations are important when using COX4I2 antibodies in Western blotting?

For optimal Western blotting results:

• Sample preparation:

  • For cell lysates: Use standard lysis buffers containing protease inhibitors

  • For tissue samples: Homogenize thoroughly and ensure complete protein extraction

• Expected molecular weight:

  • Calculated molecular weight: 20 kDa

  • Observed molecular weight: 17-18 kDa

  • Be aware of this discrepancy when analyzing your blots

• Antibody dilution and incubation:

  • Recommended dilution range: 1:500-1:3000

  • For monoclonal antibodies like clone 2E8: 1:1000-1:2000

  • Incubation time: 2 hours at room temperature or overnight at 4°C

• Controls and validation:

  • Positive control: Include HepG2 or MCF-7 cell lysates

  • Specificity control: Consider using an immunizing peptide blocking experiment to confirm specificity (as shown with ab70112)

How can COX4I2 antibodies be used to investigate tumor vascularity and blood supply?

Research has established COX4I2 as a biomarker for blood supply in adrenal tumors . To investigate this relationship:

• Experimental approach:

  • Compare COX4I2 expression between tumors with different vascularity (e.g., adrenocortical adenoma vs. pheochromocytoma)

  • Correlate expression with clinical parameters of blood supply (contrast-enhanced CT values, intraoperative blood loss)

• Methodology:

  • Quantify COX4I2 expression using both qPCR for mRNA and IHC for protein expression

  • Score IHC results based on staining intensity (0-3 scale)

  • Analyze correlation with CT enhancement values of arterial phase

• Key findings to consider:

  • COX4I2 expression was 39.14 times higher in pheochromocytoma (rich blood supply) compared to adrenocortical adenoma (poor blood supply)

  • Protein expression showed significant differences between tumor types (average intensity grade: 1.13±0.61 in ACA vs. 2.13±0.55 in PCC, P<0.0001)

  • COX4I2 expression positively correlates with CT values (r=0.611, P<0.0001), intraoperative blood loss, and operation time

  • Expression correlates with angiogenesis-related genes EPAS1, VEGFA and KDR

What experimental approaches can be used to study the role of COX4I2 in oxygen sensing and cellular hypoxic response?

To investigate COX4I2's role in oxygen sensing:

• Genetic manipulation approaches:

  • CRISPR-Cas9 mediated knockout of COX4I1 and COX4I2

  • Creation of cell lines with exclusive expression of either COX4I1 or COX4I2 isoform

  • Site-directed mutagenesis to modify specific cysteine residues (positions 40, 54, and 108) unique to COX4I2

• Functional assessments:

  • Measure oxygen affinity through determination of p50 values in cells expressing different COX4 isoforms

  • Assess mitochondrial respiration rates under varying oxygen conditions

  • Evaluate ROS production using fluorescent probes

  • Analyze NADH pool oxidation to assess metabolic changes

• Key findings from previous research:

  • The p50 value was increased twofold in COX4I2 versus COX4I1 cells, indicating decreased oxygen affinity of COX4I2-containing enzyme

  • COX4I2-expressing cells showed increased preference for mitochondrial ATP production, increased NADH pool oxidation, and lower ROS in normoxia

  • COX4I2 is essential for acute hypoxic pulmonary vasoconstriction and likely involved in oxygen sensing by carotid bodies

How can COX4I2 antibodies be used to investigate the role of COX4I2 in cell death mechanisms like ferroptosis and apoptosis?

Recent research has implicated COX4I2 in cell death pathways . To study this relationship:

• Experimental design:

  • Knockdown COX4I2 using shRNA or siRNA approaches

  • Compare cell viability, ROS levels, and markers of ferroptosis and apoptosis between control and COX4I2-deficient cells

  • Evaluate the effect of specific stimuli (such as viral infection) on these parameters

• Assessment methods:

  • Measure cell viability using MTT or similar assays

  • Quantify ROS levels using fluorescent probes

  • Assess oxidative injury markers including malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH)

  • Evaluate apoptosis markers (caspase activation, PARP cleavage)

  • Measure ferroptosis markers (lipid peroxidation, iron levels)

• Signaling pathway analysis:

  • Investigate the ERK signaling pathway activation through phosphorylation status

  • Use specific inhibitors to confirm pathway involvement

What are common challenges when using COX4I2 antibodies and how can they be addressed?

How can researchers distinguish between COX4I1 and COX4I2 when both isoforms are present in the same sample?

Differentiating between these closely related isoforms requires careful methodology:

• Isoform-specific antibodies:

  • Use antibodies specifically raised against unique peptide sequences of each isoform

  • Verify antibody specificity using knockout or knockdown controls

  • For COX4I2, use antibodies targeting the C-terminal region which differs from COX4I1

• mRNA analysis:

  • Design PCR primers that target non-homologous regions of the two isoforms

  • Use qPCR to quantify relative expression levels

  • Consider RNA-seq for comprehensive isoform profiling

• Experimental validation:

  • When performing functional studies, create cell models with exclusive expression of each isoform (as in the CRISPR-Cas9 COX4I1/2 knockout with isoform-specific knock-in approach)

  • Use these models as controls to verify antibody specificity and for comparative functional studies

• Molecular weight considerations:

  • While both isoforms have similar calculated molecular weights, small differences in migration pattern might be detected using high-percentage (15-18%) SDS-PAGE gels

  • Consider 2D gel electrophoresis for improved separation

What methodological considerations are important when studying COX4I2 under hypoxic conditions?

For research investigating COX4I2 under hypoxia:

• Hypoxia system selection:

  • Chemical hypoxia mimetics (CoCl2, DMOG) may not fully recapitulate true hypoxia effects

  • Dedicated hypoxia chambers with controlled O2 levels are preferred

  • Consider intermittent versus chronic hypoxia models, as they may yield different results

• Antibody validation under hypoxic conditions:

  • Verify antibody performance in hypoxic samples, as protein modifications or conformational changes may affect epitope accessibility

  • Include appropriate hypoxia markers (HIF-1α, VEGF) as positive controls

• Oxygen level considerations:

  • Different tissues normally experience different oxygen tensions

  • Match experimental O2 levels to physiologically relevant conditions for your tissue of interest

  • Consider measuring p50 values as demonstrated in previous research

• Time-course analysis:

  • COX4I2 expression changes may be dynamic over time in hypoxia

  • Design experiments to capture both early and late responses

  • Include multiple time points (6, 12, 24, 48 hours) to fully characterize the response

How can COX4I2 antibodies be utilized to explore its potential role as a therapeutic target?

COX4I2's involvement in tumor blood supply and cell death mechanisms suggests potential therapeutic applications:

• Target validation studies:

  • Use COX4I2 antibodies to evaluate expression levels across tumor samples and correlate with clinical outcomes

  • Perform IHC on tissue microarrays to assess COX4I2 as a prognostic biomarker

  • Combine with markers of angiogenesis (VEGF, CD31) to understand relationship with tumor vasculature

• Functional screening approaches:

  • Develop cell-based assays with COX4I2 antibodies to screen for compounds that modulate its expression or function

  • Use proximity ligation assays to identify proteins interacting with COX4I2 that might be more druggable targets

  • Evaluate effects of COX4I2 modulation on tumor response to anti-angiogenic therapies

• Therapeutic monitoring:

  • Explore whether COX4I2 levels could serve as biomarkers for treatment response in tumors

  • Investigate if changes in COX4I2 expression correlate with development of resistance to anti-angiogenic therapies

What are the technical considerations when using COX4I2 antibodies in multiparametric analyses?

For complex experimental approaches combining multiple markers:

• Multiplex immunofluorescence considerations:

  • Select antibodies raised in different host species to avoid cross-reactivity

  • When using multiple rabbit antibodies, consider sequential staining with tyramide signal amplification

  • Validate spectral overlap and bleed-through with single-stained controls

  • Include unstained controls and FMO (fluorescence minus one) controls

• Co-immunoprecipitation approaches:

  • Use appropriate controls (IgG control, input lysate)

  • Consider native versus crosslinked co-IP approaches depending on interaction strength

  • Optimized lysis conditions to preserve protein-protein interactions (milder detergents like 0.5% NP-40)

  • For COX4I2, use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Flow cytometry applications:

  • Optimize fixation and permeabilization for intracellular/mitochondrial targets

  • Use appropriate compensation controls

  • Consider the need for mitochondrial isolation or enrichment procedures

  • Recommended antibody dilution: 1:100-1:200 for flow cytometry

How does COX4I2 function differ across tissue and cell types, and what methodological approaches can address this heterogeneity?

To investigate tissue-specific functions:

• Expression profiling across tissues:

  • Use antibody panels on tissue microarrays to assess expression patterns

  • Combine with transcriptomic data to correlate protein and mRNA levels

  • Consider single-cell approaches to identify cell-type specific expression

• Functional differences by tissue:

  • Compare oxygen kinetics parameters (p50) in different cell types expressing COX4I2

  • Investigate if post-translational modifications differ between tissues

  • Evaluate isoform switching mechanisms across different cell types

• Methodological considerations:

  • Use tissue-specific knockout models rather than global knockouts

  • Consider conditional expression systems to study temporal aspects

  • Develop co-culture systems to study cell-type interactions mediated by COX4I2