CBR1 PAT2D6AT Antibody

What is CBR1 and why is it a significant research target?

CBR1 functions as an NADPH-dependent reductase with remarkably broad substrate specificity. It catalyzes the reduction of various carbonyl compounds including quinones, prostaglandins, and menadione, as well as a wide range of xenobiotics. Of particular significance to cancer research, CBR1 catalyzes the reduction of antitumor anthracyclines (doxorubicin and daunorubicin) to cardiotoxic compounds (doxorubicinol and daunorubicinol) . It also plays roles in prostaglandin metabolism, converting prostaglandin E to prostaglandin F2-alpha, and participates in glucocorticoid metabolism by catalyzing the NADPH-dependent conversion of cortisol/corticosterone to 20beta-dihydrocortisol or 20beta-corticosterone . These diverse functions make CBR1 a critical target for studies involving drug metabolism, cancer therapeutics, and endocrine research.

What types of CBR1 antibodies are available and how do they differ in research applications?

Several types of CBR1 antibodies are available for research, primarily differing in their clonality and host species:

Polyclonal antibodies typically offer higher sensitivity by recognizing multiple epitopes, while monoclonal antibodies provide greater specificity by targeting a single epitope. The choice between these types depends on your specific experimental needs and the balance required between sensitivity and specificity.

What are the validated applications for CBR1 antibodies?

CBR1 antibodies have been validated for multiple applications across different research platforms:

• Western Blot (WB): All three examined antibodies are validated for WB, detecting CBR1 at approximately 30-36 kDa .

• Immunohistochemistry (IHC): Validated for both paraffin-embedded (IHC-P) and frozen sections .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Particularly effective with recombinant monoclonal antibodies .

• Flow Cytometry: Validated with premium antibodies like the Picoband® series .

• ELISA: Some antibodies have been specifically validated for this application .

When selecting an antibody for your specific application, prioritize those with validation data in your experimental system and application of interest.

How should I optimize Western blotting protocols specifically for CBR1 detection?

For optimal CBR1 detection by Western blot, consider these methodology-specific adjustments:

• Sample Preparation: Based on validated protocols, use 30 μg of protein lysate per lane under reducing conditions .

• Gel Concentration: A 5-20% gradient SDS-PAGE gel provides optimal separation of the ~30 kDa CBR1 protein .

• Transfer Parameters: Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes for efficient protein transfer .

• Blocking Conditions: Block membranes with 5% non-fat milk in TBS for 1.5 hours at room temperature to minimize background .

• Antibody Concentration: For primary antibody incubation, use approximately 0.5 μg/mL and incubate overnight at 4°C for optimal signal-to-noise ratio .

• Detection System: Enhanced Chemiluminescent (ECL) detection systems provide excellent sensitivity for CBR1 visualization .

These parameters have been specifically validated with human cell lines (SW620, SK-OV-3, 293T, HeLa, MCF-7, SH-SY5Y), rat testis tissue, and mouse liver tissue, showing consistent detection of CBR1 .

What are the critical considerations for CBR1 antibody-based immunohistochemistry?

For successful IHC detection of CBR1:

• Antigen Retrieval: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is crucial for optimal epitope exposure in paraffin-embedded tissues .

• Blocking Parameters: Block tissue sections with 10% goat serum to minimize non-specific binding .

• Antibody Concentration: Use approximately 2 μg/ml of primary antibody with overnight incubation at 4°C for best results .

• Secondary Detection: Peroxidase-conjugated secondary antibodies with 30-minute incubation at 37°C provide good signal development .

• Chromogen Selection: DAB (3,3'-diaminobenzidine) has been validated as an effective chromogen for CBR1 visualization in various tissue types .

These protocols have been specifically validated in human breast cancer, liver cancer, lung cancer, and rectal cancer tissues, as well as mouse kidney tissue, demonstrating the versatility of CBR1 antibodies across different tissue types .

How can I validate the specificity of my CBR1 antibody?

Validating antibody specificity is crucial for reliable results. Implement these methodological approaches:

• Positive Control Selection: Use tissues or cell lines known to express CBR1, such as human SW620, SK-OV-3, 293T, HeLa, MCF-7, SH-SY5Y cell lines, rat testis tissue, or mouse liver tissue .

• Western Blot Validation: Confirm a single band at the expected molecular weight of ~30 kDa .

• Knockout/Knockdown Controls: Compare signal between wild-type samples and CBR1 knockout or knockdown samples.

• Peptide Competition Assay: Pre-incubate the antibody with purified CBR1 protein or immunogen peptide to block specific binding.

• Cross-Reactivity Assessment: Test the antibody against related proteins to ensure specificity.

• Multiple Antibody Approach: Use different antibodies targeting different epitopes of CBR1 to confirm consistent localization and expression patterns.

How do I address non-specific binding when using CBR1 antibodies?

Non-specific binding can significantly impact experimental outcomes. Implement these methodological solutions:

• Optimize Blocking Conditions: Extend blocking time to 2 hours or test alternative blocking agents such as BSA or normal serum from the secondary antibody host species.

• Titrate Antibody Concentration: Perform a dilution series to identify the optimal concentration that maximizes specific signal while minimizing background.

• Adjust Incubation Parameters: Reduce primary antibody incubation temperature (4°C instead of room temperature) and extend incubation time.

• Increase Wash Stringency: Add 0.1% Tween-20 to wash buffers and increase both the number and duration of washes.

• Secondary Antibody Selection: Use highly cross-adsorbed secondary antibodies to reduce cross-reactivity with endogenous immunoglobulins.

• Pre-adsorption: Pre-adsorb the primary antibody with proteins from the species being tested to remove cross-reactive antibodies.

What controls should be incorporated when working with CBR1 antibodies?

Proper controls are essential for experimental validity:

Implementing these controls systematically will significantly enhance the reliability and interpretability of your CBR1 antibody-based experimental results.

How can CBR1 antibodies be employed in cancer research studies?

CBR1 has significant implications in cancer therapeutics, particularly regarding anthracycline metabolism:

• Chemoresistance Studies: Use CBR1 antibodies to investigate correlation between CBR1 expression levels and resistance to anthracycline-based chemotherapies.

• Cardiotoxicity Assessment: Examine CBR1 expression in cardiac tissues to study the relationship between CBR1 activity and anthracycline-induced cardiotoxicity.

• Biomarker Evaluation: Assess CBR1 as a potential prognostic or predictive biomarker through IHC analysis of patient tumor samples.

• Co-localization Studies: Combine CBR1 antibodies with markers of drug efflux pumps or other resistance mechanisms using multiplex immunofluorescence.

• Tumor Microenvironment Analysis: Investigate CBR1 expression across different cell types within the tumor microenvironment.

The validated application of CBR1 antibodies in human breast cancer, liver cancer, lung cancer, and rectal cancer tissues makes them particularly valuable tools for translational cancer research .

What methodological approaches are recommended for studying CBR1's role in drug metabolism?

To effectively investigate CBR1's role in drug metabolism:

• Expression Correlation Studies: Use Western blotting to correlate CBR1 expression levels with metabolite formation in various tissue types.

• Subcellular Localization: Employ immunofluorescence with organelle markers to determine the subcellular distribution of CBR1 in metabolically active tissues.

• Tissue Distribution Mapping: Apply IHC to map CBR1 expression across different organs involved in drug metabolism (liver, kidney, intestine).

• Patient Sample Analysis: Analyze CBR1 expression in patient samples to correlate with drug response and toxicity profiles.

• Intervention Studies: Combine CBR1 antibody detection with inhibitor studies or genetic manipulation to establish causative relationships.

How should I interpret contradictory results between different CBR1 antibodies?

When faced with contradictory results:

• Review Epitope Information: Determine if the antibodies recognize different epitopes, which might explain differential detection based on protein conformation or post-translational modifications.

• Compare Validated Applications: Check if each antibody has been specifically validated for your application of interest.

• Assess Clonality Differences: Consider whether discrepancies might result from using monoclonal versus polyclonal antibodies.

• Evaluate Specificity Data: Review each antibody's specificity validation data and determine which has the most robust validation.

• Repeat with Additional Controls: Incorporate additional controls such as knockdown/knockout samples or peptide competition assays.

• Consult Literature: Compare your findings with published results using the same antibodies.

• Consider Alternative Detection Methods: Validate your findings using non-antibody-based methods such as mass spectrometry or mRNA analysis.

What quantification methods are most appropriate for CBR1 Western blot analysis?

For reliable quantification:

• Densitometric Analysis: Use specialized software (ImageJ, Image Lab, etc.) to perform densitometric analysis of CBR1 bands.

• Normalization Strategy: Always normalize CBR1 signal to appropriate loading controls (β-actin, GAPDH, total protein) to account for loading variations.

• Linear Range Determination: Establish the linear range of detection for both CBR1 and your loading control to ensure quantification within this range.

• Technical Replicates: Perform at least three independent experiments for statistical validity.

• Standard Curve Inclusion: Consider including a standard curve using recombinant CBR1 protein for absolute quantification.

• Statistical Analysis: Apply appropriate statistical tests (t-test, ANOVA) based on your experimental design.