CBR1 Antibody

What is CBR1 and why is it important in pharmacological studies?

CBR1 (Carbonyl reductase 1) is a cytosolic short-chain dehydrogenase that catalyzes the NADPH-dependent reduction of a wide variety of carbonyl compounds. It has significant pharmacological importance due to its role in metabolizing several therapeutic drugs, particularly anticancer anthracyclines like doxorubicin and daunorubicin . CBR1 converts these drugs into cardiotoxic metabolites (doxorubicinol and daunorubicinol), which may contribute to treatment-related cardiotoxicity. Therefore, understanding CBR1 expression and activity is crucial when studying anthracycline pharmacodynamics and developing strategies to mitigate cardiotoxicity in cancer treatment .

Additionally, CBR1 participates in glucocorticoid metabolism by catalyzing the NADPH-dependent reduction of cortisol/corticosterone into 20beta-dihydrocortisol or 20beta-corticosterone, which function as weak agonists of nuclear receptors NR3C1 and NR3C2 in adipose tissue . This versatile enzyme also influences lipid metabolism, hormone synthesis, and the reduction of various xenobiotics, making it a target of interest in multiple research fields .

What are the most reliable antibodies available for CBR1 detection?

Based on the available literature and product information, several validated antibodies have demonstrated reliability for CBR1 detection across different applications:

• Rabbit Recombinant Monoclonal CBR1 antibody [EPR9660] (ab156590) - This antibody has been validated for immunohistochemistry on paraffin sections (IHC-P), western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF) applications with human samples. It has been cited in scientific publications, indicating peer validation .

• Rabbit Polyclonal CBR1 antibody (ab186825) - This antibody has been validated for western blotting and demonstrates reactivity with both human and mouse samples. It has been cited in three scientific publications, suggesting reliable performance in research settings .

• Anti-CBR1 Picoband® Antibody (A02825-1) - This rabbit host antibody has been validated across multiple applications including ELISA, flow cytometry, immunofluorescence, immunohistochemistry, immunocytochemistry, and western blotting. It demonstrates reactivity with human, mouse, and rat samples .

When selecting an antibody, researchers should consider the specific application requirements, species reactivity needed, and whether monoclonal specificity or polyclonal broader epitope recognition would be more suitable for their experimental design.

What is the relationship between CBR1 expression and anthracycline drug metabolism?

CBR1 plays a pivotal role in anthracycline drug metabolism through its NADPH-dependent reductase activity. The enzyme catalyzes the reduction of anthracycline drugs doxorubicin and daunorubicin to their respective alcohol metabolites, doxorubicinol and daunorubicinol . This metabolic conversion has significant clinical implications as these alcohol metabolites exhibit reduced antitumor efficacy while possessing increased cardiotoxicity compared to their parent compounds.

Studies have shown that variability in CBR1 expression and activity between individuals may contribute to the unpredictable pharmacodynamics of doxorubicin treatment . Research examining liver samples has demonstrated up to 8-fold variation in CBR1 protein expression among black donors and 6-fold variation among white donors, suggesting substantial inter-individual differences in anthracycline metabolism capacity .

The relationship between CBR1 expression and anthracycline metabolism has important implications for personalized medicine approaches in cancer treatment, as patients with higher CBR1 expression might experience enhanced conversion to cardiotoxic metabolites, potentially requiring adjusted dosing strategies or cardioprotective interventions.

How can CBR1 antibodies be optimized for quantitative protein expression analysis in tissue samples?

For precise quantitative analysis of CBR1 protein expression in tissue samples, researchers should implement a comprehensive optimization strategy:

Quantitative Immunoblotting Protocol:

• Sample preparation: Prepare cytosolic fractions from tissue samples (as demonstrated with liver cytosols in the literature), typically using 150 μg of protein per sample .

• Standard curve generation: Prepare a calibration curve using purified recombinant CBR1 protein at multiple concentrations (e.g., 0.05, 0.08, 0.10, 0.15, 0.20, and 0.30 μg) .

• Gel separation: Separate samples on 4-10% gradient gels at controlled voltage (e.g., 90V for 90 minutes) .

• Transfer optimization: Transfer proteins to PVDF membranes under standardized conditions to ensure consistent protein transfer across all samples.

• Blocking protocol: Block membranes with appropriate blocking buffer (e.g., StartingBlock T20) for 1 hour at room temperature .

• Antibody incubation: Incubate with validated anti-CBR1 antibody at optimized dilution (typically 1:2000 for the polyclonal antibody) .

• Detection system calibration: Use an enhanced chemiluminescence system with linear detection range and capture images using a calibrated documentation system.

• Normalization strategy: Include β-actin or other housekeeping protein detection for sample normalization .

• Quantification: Calculate CBR1 protein concentration by extrapolation from the standard curve, expressing results in standardized units (e.g., nmol/g cytosolic protein) .

This approach can achieve reliable quantification with acceptable limits of detection (0.01 μg) and quantification (0.02 μg), with a linear detection range of 0.05–0.30 μg and coefficient of variation of approximately 9.5%, based on published methodology .

What strategies should be employed to validate CBR1 antibody specificity across different experimental systems?

Validating CBR1 antibody specificity requires a multi-faceted approach to ensure reliable research outcomes:

Comprehensive Validation Strategy:

• Sequence homology assessment: Evaluate potential cross-reactivity with related proteins, particularly CBR3, which shares structural similarities with CBR1. Published research has demonstrated that well-validated anti-CBR1 antibodies show no immunoreactive bands when tested against purified recombinant CBR3 protein .

• Knockout/knockdown controls: Utilize CRISPR/Cas9-edited cell lines or siRNA-mediated knockdown of CBR1 to create negative controls for antibody validation.

• Overexpression systems: Generate cells overexpressing tagged CBR1 to confirm antibody detection at the expected molecular weight (~30 kDa).

• Peptide competition assays: Pre-incubate the antibody with excess immunizing peptide or recombinant CBR1 protein before application to verify signal specificity.

• Multi-technique confirmation: Validate antibody performance across complementary techniques (Western blot, immunohistochemistry, immunofluorescence) to ensure consistent results.

• Species cross-reactivity verification: Test antibody performance across target species using appropriate positive controls, especially important when working with model organisms.

• Batch-to-batch validation: Implement standardized validation protocols for each new antibody lot to ensure consistent performance over time.

The literature demonstrates successful application of these strategies, with researchers confirming single-band detection at approximately 30 kDa for CBR1, appropriate sample integrity verification using housekeeping proteins, and absence of cross-reactivity with the related CBR3 protein .

How can genetic polymorphisms in CBR1 affect antibody-based detection methods?

Genetic polymorphisms in CBR1 can significantly impact antibody-based detection methods, requiring careful consideration in experimental design:

Impact of CBR1 Polymorphisms on Detection:

The CBR1 gene harbors several known polymorphisms that may influence antibody binding and detection efficacy. Key considerations include:

• Epitope alterations: Nonsynonymous SNPs like CBR1 V88I (rs1143663) modify the amino acid sequence, potentially altering antibody epitopes and affecting binding affinity . Antibodies targeting regions containing such variations may show differential binding depending on the subject's genotype.

• Expression-modifying polymorphisms: The 3′-UTR polymorphism 1096G>A appears to influence CBR1 expression levels, with homozygous G/G genotypes showing a trend toward higher CBR1 mRNA levels compared to heterozygous G/A genotypes . This variation in expression could impact quantitative analyses if not properly accounted for.

• Population-specific considerations: Some CBR1 polymorphisms show ethnicity-dependent distribution patterns. For example, the V88I variant appears primarily in individuals with African ancestry (q = 0.014) , while the 1096G>A polymorphism was detected in samples from white donors . These population differences necessitate careful subject selection and stratification in comparative studies.

• Protocol adjustments: When working with genetically diverse samples, researchers should consider:

  • Genotyping subjects to stratify samples by CBR1 genetic variants

  • Selecting antibodies targeting conserved epitopes when possible

  • Using multiple antibodies targeting different epitopes to ensure detection regardless of variant status

  • Correlating antibody detection signals with functional enzyme activity assays to validate findings

Research has demonstrated that CBR1 1096G>A genotype status correlates with both CBR1 protein levels and enzymatic activity, highlighting the importance of considering genetic background when interpreting antibody-based detection results .

What are the best techniques for analyzing CBR1 expression at both mRNA and protein levels?

Comprehensive analysis of CBR1 expression requires coordinated assessment at both mRNA and protein levels using optimized techniques:

mRNA Level Analysis:

• Quantitative real-time RT-PCR (qRT-PCR): The literature demonstrates successful CBR1 mRNA quantification using this approach, with careful attention to:

  • RNA extraction quality (high RIN values)

  • Specific primer design to avoid amplification of related family members

  • Appropriate housekeeping gene selection (β-actin has been validated)

  • Normalization to a reference sample for relative quantification

• RNA sequencing: For broader transcriptomic analysis, RNA-seq provides comprehensive CBR1 expression data alongside global gene expression patterns, enabling correlation with regulatory networks.

Protein Level Analysis:

• Quantitative immunoblotting: Using recombinant CBR1 protein standards allows precise quantification of CBR1 protein levels, as demonstrated in liver cytosols where expression has been reported in the range of 2.2–19.2 nmol/g cytosolic protein .

• Immunohistochemistry (IHC): For tissue localization studies, validated anti-CBR1 antibodies like the Rabbit Recombinant Monoclonal CBR1 antibody [EPR9660] have demonstrated reliability in IHC-P applications .

• Immunofluorescence (IF): For subcellular localization and co-localization studies, antibodies validated for IF applications enable visualization of CBR1 distribution within cells .

• Flow cytometry: For single-cell analysis in heterogeneous populations, flow cytometry using validated anti-CBR1 antibodies allows quantification of expression levels across different cell populations .

Integrated Analysis Approach:

Research has shown that CBR1 mRNA and protein levels may not always correlate directly , highlighting the importance of analyzing both levels to fully understand CBR1 regulation. A comprehensive approach combining qRT-PCR for mRNA quantification with immunoblotting for protein quantification provides the most complete picture of CBR1 expression patterns.

What controls and validation steps are essential when using CBR1 antibodies in western blotting applications?

Implementing robust controls and validation steps is critical for reliable CBR1 detection in western blotting applications:

Essential Controls and Validation Steps:

• Positive controls:

  • Recombinant human CBR1 protein at known concentrations (0.05-0.30 μg range) for standard curve generation

  • Well-characterized cell lines or tissue samples known to express CBR1 (liver tissue is particularly suitable as a positive control)

• Negative controls:

  • CBR1 knockout or knockdown samples where available

  • Tissues known to express minimal CBR1

  • Pre-absorption of antibody with immunizing peptide/recombinant protein

• Specificity validation:

  • Testing for cross-reactivity with related proteins, particularly CBR3 (research has confirmed absence of cross-reactivity between well-validated CBR1 antibodies and recombinant CBR3)

  • Verification of expected molecular weight (approximately 30 kDa for human CBR1)

• Loading controls:

  • β-actin detection for normalization and sample integrity confirmation

  • Total protein staining methods (Ponceau S, SYPRO Ruby) for alternative normalization

• Technical validation:

  • Antibody dilution optimization (typically 1:2000 for polyclonal antibodies)

  • Linearity testing across a range of protein loads

  • Detection system calibration to ensure measurements within linear range

  • Replicate analysis to establish coefficient of variation (published methods achieved CV = 9.5%)

• Quantification standards:

  • Inclusion of recombinant CBR1 standards for absolute quantification

  • Defined limits of detection (0.01 μg) and quantification (0.02 μg)

Following these validation steps ensures generation of reliable and reproducible data when using CBR1 antibodies for western blotting applications.

How should researchers interpret contradictory results between CBR1 mRNA expression and protein levels?

Discrepancies between CBR1 mRNA and protein expression levels are not uncommon and require careful interpretation:

Interpretation Framework for Contradictory Results:

• Post-transcriptional regulation mechanisms:

  • MicroRNA regulation: CBR1 mRNA may be targeted by specific microRNAs that regulate translation efficiency without affecting mRNA stability

  • RNA-binding proteins: Regulatory proteins might alter CBR1 mRNA translation rates

  • 3′-UTR polymorphisms: The 1096G>A polymorphism in the CBR1 3′-UTR may influence translation efficiency or mRNA stability

• Post-translational regulation factors:

  • Protein stability differences: CBR1 protein may have variable half-life depending on cellular conditions or genetic background

  • Proteasomal degradation: Variations in ubiquitination and degradation pathways could affect steady-state protein levels

  • Post-translational modifications: Phosphorylation or other modifications may influence antibody recognition or protein stability

• Technical considerations:

  • Antibody recognition efficiency: Epitope accessibility or post-translational modifications could affect antibody binding

  • Sample preparation differences: Differential extraction efficiency between RNA and protein isolation methods

  • Normalization strategies: Different reference genes/proteins used for normalization may introduce variability

• Biological variability:

  • Temporal dynamics: mRNA and protein may reflect different time points in the expression process

  • Tissue-specific factors: Liver samples have shown up to 8-fold variation in CBR1 protein levels between individuals

  • Genetic background influence: CBR1 1096G>A genotype status has been associated with protein levels and enzymatic activity

• Recommended analytical approach:

  • Correlate with functional assays: Measure CBR1 enzymatic activity (e.g., doxorubicinol production rates) to determine functional relevance

  • Examine genotype status: Determine CBR1 polymorphism status to account for genetically-driven expression differences

  • Perform time-course studies: Examine both mRNA and protein at multiple time points to capture expression dynamics

Research has demonstrated that correlation analysis between CBR1 mRNA and protein levels may not show significant association , underscoring the importance of multi-level analysis when studying CBR1 expression.

How can CBR1 antibodies be utilized to study anthracycline-induced cardiotoxicity mechanisms?

CBR1 antibodies provide valuable tools for investigating the mechanisms of anthracycline-induced cardiotoxicity through several strategic applications:

Research Applications for Cardiotoxicity Studies:

• Tissue-specific expression profiling:

  • Immunohistochemistry of cardiac tissue to quantify CBR1 expression in cardiomyocytes

  • Comparative analysis of CBR1 expression across different cardiac regions (atria, ventricles) to identify susceptible areas

  • Correlation of expression patterns with sites of anthracycline-induced damage

• Cellular response monitoring:

  • Western blotting to track changes in CBR1 expression levels following anthracycline exposure

  • Immunofluorescence to examine subcellular localization changes during drug metabolism

  • Flow cytometry to quantify CBR1 expression in isolated cardiomyocytes from treated vs. untreated models

• Mechanistic pathway studies:

  • Co-immunoprecipitation with CBR1 antibodies to identify protein interaction partners in cardiac tissue

  • Chromatin immunoprecipitation (ChIP) using transcription factor antibodies to study CBR1 regulation

  • Proximity ligation assays to investigate protein-protein interactions involving CBR1 in situ

• Genetic association validation:

  • Immunoblotting to quantify CBR1 protein in cardiac samples from individuals with different CBR1 genotypes

  • Correlation of protein levels with functional assays measuring the conversion of doxorubicin to doxorubicinol

  • Association of CBR1 expression patterns with clinical cardiotoxicity outcomes

• Intervention evaluation:

  • Monitoring changes in CBR1 expression/activity in response to cardioprotective agents

  • Evaluating the impact of CBR1 inhibitors on anthracycline metabolism and cardiac function

  • Assessing the efficacy of targeted approaches to modulate CBR1 activity specifically in cardiac tissue

The critical role of CBR1 in converting anthracyclines to cardiotoxic alcohol metabolites (doxorubicinol and daunorubicinol) makes it an essential target for understanding the variable susceptibility to anthracycline-induced cardiotoxicity among cancer patients .

What experimental design considerations are important when studying CBR1 genetic polymorphisms and their effect on drug metabolism?

When investigating CBR1 genetic polymorphisms and their impact on drug metabolism, researchers should implement a comprehensive experimental design that addresses several critical factors:

Experimental Design Framework:

• Population selection and stratification:

  • Include diverse ethnic backgrounds to capture polymorphism variation (e.g., CBR1 V88I appears primarily in individuals with African ancestry)

  • Verify genetic ancestry using appropriate markers to ensure accurate ethnicity classification

  • Properly power studies to detect polymorphism effects (consider frequency of variants like 1096G>A with reported allele frequencies of p = 0.875; q = 0.125)

• Comprehensive genotyping approach:

  • Sequence the entire CBR1 gene including promoter, introns, and 3'-UTR regions to identify all relevant polymorphisms

  • Include known functional variants (1096G>A, V88I) as well as novel polymorphisms

  • Consider genotyping related enzymes involved in anthracycline metabolism for pathway-wide analysis

• Multi-level expression analysis:

  • Quantify mRNA expression using qRT-PCR with appropriate normalization

  • Measure protein levels using validated antibodies and quantitative immunoblotting

  • Determine enzyme activity through specific substrate metabolism (e.g., doxorubicin to doxorubicinol conversion)

  • Correlate genotype with all three levels (mRNA, protein, activity) to identify functional impacts

• Tissue relevance:

  • Focus on pharmacologically relevant tissues (liver for metabolism, cardiac tissue for toxicity endpoints)

  • Consider tissue-specific expression patterns and regulation mechanisms

  • Use appropriate in vitro models that recapitulate in vivo metabolism

• Functional validation:

  • Employ site-directed mutagenesis to recreate polymorphisms in expression systems

  • Use CRISPR/Cas9 to introduce or correct polymorphisms in cell models

  • Develop transgenic animal models expressing human CBR1 variants when appropriate

• Clinical correlation:

  • Design translational studies that connect polymorphism status with clinical outcomes

  • Account for other contributing factors (concomitant medications, comorbidities)

  • Consider pharmacokinetic parameters alongside CBR1 genetic status

Research has demonstrated that CBR1 1096G>A genotype status associates with both CBR1 protein levels and enzyme activity (measured as the rate of doxorubicinol synthesis), highlighting the importance of connecting genetic variation to functional outcomes .

How can CBR1 antibodies contribute to developing improved cancer therapeutics with reduced cardiotoxicity?

CBR1 antibodies represent valuable tools in the development of cancer therapeutics with improved safety profiles, particularly for reducing anthracycline-associated cardiotoxicity:

Strategic Applications in Drug Development:

• Target validation and biomarker development:

  • Quantitative immunoblotting to establish baseline CBR1 expression in patient populations

  • Correlation of pre-treatment CBR1 levels with subsequent cardiotoxicity risk

  • Development of companion diagnostics to identify patients at elevated risk who might benefit from alternative treatments or cardioprotective strategies

• Drug screening and evaluation:

  • High-throughput screening assays incorporating CBR1 antibodies to identify compounds that modulate CBR1 activity

  • Evaluation of lead compounds' effects on CBR1 expression in cardiac tissue

  • Assessment of combination therapies targeting both cancer cells and CBR1-mediated cardiotoxicity pathways

• Mechanistic investigations:

  • Immunoprecipitation to identify protein-protein interactions that could be targeted to modulate CBR1 activity

  • ChIP-seq applications to map transcriptional regulation of CBR1 and identify potential intervention points

  • Proteomics approaches to characterize CBR1 post-translational modifications that influence enzyme activity

• Drug delivery optimization:

  • Monitoring CBR1 expression in target vs. non-target tissues to optimize drug delivery strategies

  • Evaluation of tissue-specific CBR1 inhibition approaches to prevent cardiotoxic metabolite formation

  • Development of nanoparticle or antibody-drug conjugate delivery systems that bypass CBR1-mediated metabolism

• Predictive model development:

  • Integration of CBR1 expression data with machine learning approaches to predict cardiotoxicity risk

  • Development of physiologically-based pharmacokinetic models incorporating individual CBR1 expression/activity data

  • Creation of in vitro cardiac models with varying CBR1 expression levels to test drug candidates

The central role of CBR1 in converting anthracyclines like doxorubicin and daunorubicin to their cardiotoxic metabolites (doxorubicinol and daunorubicinol) makes it a prime target for interventions aimed at reducing treatment-related cardiotoxicity while maintaining anticancer efficacy .

What are the optimal sample preparation methods for different CBR1 antibody applications?

Optimal sample preparation is critical for successful CBR1 antibody applications across different experimental techniques:

Technique-Specific Sample Preparation:

• Western Blotting:

  • Tissue samples: Prepare cytosolic fractions through differential centrifugation (liver cytosols have been successfully used at 150 μg per lane)

  • Cell samples: Lyse cells in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors

  • Protein denaturation: Heat samples in a boiling water bath for 5 minutes with Laemmli buffer and 5% β-mercaptoethanol

  • Gel selection: Use 4-10% gradient gels for optimal CBR1 separation

  • Loading control: Include β-actin detection for normalization

• Immunohistochemistry (IHC):

  • Fixation: Use 10% neutral buffered formalin for optimal epitope preservation

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Blocking: Apply appropriate blocking solution (e.g., StartingBlock T20) to minimize background

  • Controls: Include positive control tissues (liver) and negative controls (antibody omission and pre-absorption)

• Immunofluorescence (IF):

  • Cell preparation: Grow cells on coated coverslips or chamber slides

  • Fixation: Use 4% paraformaldehyde (10-15 minutes) followed by permeabilization with 0.1% Triton X-100

  • Blocking: Incubate with 5% normal serum from the same species as the secondary antibody

  • Nuclear counterstain: Apply DAPI to visualize nuclei

• Flow Cytometry:

  • Cell preparation: Create single-cell suspensions through gentle mechanical dissociation

  • Fixation/permeabilization: Use commercial kits designed for intracellular antigen detection

  • Controls: Include unstained cells, isotype controls, and known positive samples

  • Viability dye: Incorporate to exclude dead cells from analysis

• Immunoprecipitation:

  • Lysis conditions: Use NP-40 or CHAPS-based buffers to maintain native protein conformation

  • Pre-clearing: Incubate lysates with protein A/G beads before antibody addition

  • Antibody coupling: Consider cross-linking antibodies to beads for cleaner results

  • Wash stringency: Optimize salt concentration to maintain specific interactions

Validated CBR1 antibodies have been successfully used across these applications, with western blotting being particularly well-documented for detecting CBR1 as a single band of approximately 30 kDa in human samples .

What are the common pitfalls in CBR1 antibody-based research and how can they be avoided?

Researchers should be aware of several common pitfalls when using CBR1 antibodies and implement appropriate strategies to avoid them:

Common Pitfalls and Mitigation Strategies:

• Cross-reactivity with related proteins:

  • Pitfall: CBR3 shares structural similarities with CBR1 and may cause false-positive results with non-specific antibodies

  • Solution: Validate antibody specificity against recombinant CBR3 protein ; published research confirms well-validated anti-CBR1 antibodies show no cross-reactivity with CBR3

• Variable CBR1 expression levels:

  • Pitfall: Up to 8-fold variation in CBR1 protein levels has been observed in human liver samples , which can complicate interpretation

  • Solution: Include appropriate positive controls and standard curves; analyze sufficient biological replicates to account for natural variation

• Genetic polymorphism effects:

  • Pitfall: CBR1 polymorphisms like 1096G>A influence protein expression and activity , potentially affecting antibody-based quantification

  • Solution: Genotype samples when possible; stratify analyses by genotype; use antibodies targeting conserved epitopes

• Post-translational modifications:

  • Pitfall: Potential modifications may alter antibody epitope accessibility

  • Solution: Use multiple antibodies targeting different regions; validate results with functional assays measuring CBR1 activity

• Non-linear signal response:

  • Pitfall: Detection systems may saturate, leading to inaccurate quantification

  • Solution: Establish linear detection range (e.g., 0.05–0.30 μg for recombinant CBR1) ; perform dilution series to ensure measurements fall within linear range

• Inconsistent sample preparation:

  • Pitfall: Variation in extraction efficiency between samples affects quantification

  • Solution: Standardize all preparation steps; use internal controls; normalize to housekeeping proteins like β-actin

• Inadequate controls:

  • Pitfall: Lacking proper positive/negative controls leads to misinterpretation

  • Solution: Include recombinant CBR1 standards, known positive samples, antibody pre-absorption controls, and isotype controls as appropriate

• Batch-to-batch antibody variation:

  • Pitfall: Different lots may have variable performance characteristics

  • Solution: Validate each new antibody lot; maintain reference samples for comparison; consider recombinant monoclonal antibodies for improved consistency

• Inappropriate application conditions:

  • Pitfall: Using non-validated antibody dilutions or incubation conditions

  • Solution: Optimize conditions for each application; follow manufacturer recommendations and published protocols (e.g., 1:2000 dilution for western blotting)

Implementing these strategies will significantly improve the reliability and reproducibility of CBR1 antibody-based research.

How can multiplexed detection approaches be used to correlate CBR1 with related metabolic enzymes?

Multiplexed detection strategies offer powerful tools for examining CBR1 in the context of related metabolic pathways:

Advanced Multiplexing Approaches:

• Multiplex immunofluorescence/immunohistochemistry:

  • Methodology: Utilize primary antibodies from different host species or directly labeled primary antibodies to simultaneously detect CBR1 and related enzymes (e.g., aldo-keto reductases, cytochrome P450s)

  • Analysis: Apply spectral unmixing algorithms to separate fluorophore signals

  • Applications: Visualize co-localization of CBR1 with other drug-metabolizing enzymes in tissue sections

  • Advantage: Preserves spatial context within tissue architecture

• Multiplex western blotting:

  • Methodology: Use antibodies with distinct species origins or target proteins with sufficient molecular weight differences

  • Approach 1: Sequential probing with stripping between antibodies

  • Approach 2: Fluorescent secondary antibodies with distinct emission spectra

  • Applications: Quantify relative expression of CBR1 alongside other NADPH-dependent reductases in the same sample

  • Advantage: Allows direct quantitative comparison of multiple targets

• Mass cytometry (CyTOF):

  • Methodology: Label antibodies with isotopically pure metals instead of fluorophores

  • Detection: Use time-of-flight mass spectrometry to identify metal tags

  • Scale: Simultaneously measure >40 parameters at single-cell resolution

  • Applications: Profile CBR1 expression across heterogeneous cell populations alongside other metabolic enzymes

  • Advantage: Eliminates spectral overlap issues encountered in conventional flow cytometry

• Protein array approaches:

  • Methodology: Develop antibody arrays containing CBR1 and related enzyme antibodies

  • Detection: Apply labeled protein samples and measure binding signals

  • Applications: Screen multiple samples for expression patterns across metabolic enzyme networks

  • Advantage: High-throughput analysis of numerous samples

• Proximity ligation assay (PLA):

  • Methodology: Use oligonucleotide-labeled secondary antibodies that generate amplifiable DNA signals when in close proximity

  • Applications: Detect protein-protein interactions between CBR1 and potential binding partners

  • Advantage: Visualize transient interactions in situ with high sensitivity

• Single-cell multi-omics:

  • Methodology: Combine antibody-based protein detection with RNA sequencing at single-cell resolution

  • Applications: Correlate CBR1 protein levels with mRNA expression of related enzymes

  • Advantage: Links transcriptional and translational regulation patterns

These multiplexed approaches enable comprehensive characterization of CBR1 in relation to other enzymes involved in drug metabolism, providing deeper insights into the coordination of metabolic pathways and potential interactions that influence anthracycline metabolism and cardiotoxicity .