CBR1 Monoclonal Antibody

What is CBR1 and what biological functions does it perform?

CBR1 (Carbonyl Reductase 1) is an NADPH-dependent reductase with broad substrate specificity that catalyzes the reduction of a wide variety of carbonyl compounds including quinones, prostaglandins, menadione, and various xenobiotics . The enzyme plays significant roles in several physiological processes including lipid metabolism and hormone synthesis . CBR1 notably catalyzes the reduction of antitumor anthracyclines such as doxorubicin and daunorubicin to cardiotoxic compounds doxorubicinol and daunorubicinol, which has important implications for cancer treatment and associated cardiotoxicity . Additionally, CBR1 participates in glucocorticoid metabolism by catalyzing the NADPH-dependent conversion of cortisol/corticosterone into 20beta-dihydrocortisol or 20beta-corticosterone, which function as weak agonists of specific nuclear receptors in adipose tissue . This multifunctional enzyme can also convert prostaglandin E to prostaglandin F2-alpha and participates in the reduction of S-nitrosoglutathione, highlighting its diverse physiological roles .

What are the typical applications for CBR1 monoclonal antibodies in research?

CBR1 monoclonal antibodies serve as valuable tools across multiple research applications focusing on protein detection and characterization. Western blotting (WB) represents one of the primary applications, allowing researchers to detect and quantify CBR1 protein expression in various tissue and cell lysates with recommended dilutions typically around 1:1000 . Immunocytochemistry (ICC) provides another crucial application, enabling the visualization of CBR1 localization within cells at typical working dilutions of approximately 1:100 . Some CBR1 antibodies are also validated for immunohistochemistry on paraffin-embedded tissues (IHC-P), allowing researchers to examine CBR1 expression patterns in tissue specimens . Additionally, certain anti-CBR1 monoclonal antibodies are suitable for enzyme-linked immunosorbent assays (ELISA) and immunofluorescence (ICC/IF) applications, expanding their utility in quantitative protein detection and co-localization studies with other cellular components . These diverse applications make CBR1 monoclonal antibodies essential tools for researching carbonyl metabolism, drug resistance mechanisms, and related physiological processes.

How should CBR1 monoclonal antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of CBR1 monoclonal antibodies are crucial for maintaining their functionality and specificity over time. For long-term storage, CBR1 monoclonal antibodies should be stored at -20°C, where they typically remain stable for up to one year . The antibodies are commonly provided in liquid form containing preservatives such as sodium azide and stabilizers like glycerol to maintain their integrity . For frequent use and short-term storage (up to one month), researchers can store the antibodies at 4°C to avoid the damaging effects of repeated freeze-thaw cycles . When handling these antibodies, it's important to minimize exposure to room temperature and to aliquot the stock solution into smaller volumes if frequent use is anticipated, as repeated freeze-thaw cycles can significantly degrade antibody performance . Many manufacturers provide their CBR1 monoclonal antibodies in formulations containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.2, which helps maintain stability during storage . Researchers should always follow manufacturer-specific storage recommendations, as formulations may vary slightly between different antibody products and suppliers.

What are the typical reactive species and cross-reactivity profiles for CBR1 monoclonal antibodies?

CBR1 monoclonal antibodies exhibit specific reactive profiles that researchers must consider when designing experiments. Most commercially available CBR1 monoclonal antibodies are primarily developed to react with human CBR1 protein, as evidenced by products like Boster Bio's M02825 and similar antibodies from other manufacturers . These antibodies are rigorously validated to ensure specificity for human CBR1 with minimal cross-reactivity to other proteins, allowing for confident interpretation of experimental results . Many manufacturers specifically test and guarantee no cross-reactivity with other proteins, which is essential for accurate protein detection and quantification . While human-reactive antibodies predominate, researchers should carefully evaluate whether a particular CBR1 antibody will recognize the species-specific CBR1 variants in their experimental system . The specificity of these antibodies is particularly important when investigating tissues or cell types where multiple carbonyl reductases or related dehydrogenases may be expressed simultaneously, as this could potentially lead to ambiguous results without proper validation . Researchers working with animal models should verify the cross-species reactivity of their selected antibody through literature searches or preliminary validation experiments.

How can researchers validate the specificity of CBR1 monoclonal antibodies in their experimental systems?

Validating the specificity of CBR1 monoclonal antibodies requires a multi-faceted approach to ensure reliable experimental outcomes. Researchers should begin with positive and negative control samples in their detection system of choice, such as lysates from cells known to express high levels of CBR1 versus those with minimal expression or CBR1 knockout models . Performing peptide competition assays represents another essential validation strategy, where pre-incubating the antibody with purified CBR1 protein or immunizing peptide should significantly reduce or eliminate the specific signal if the antibody is truly selective for CBR1 . Many manufacturers validate their antibodies using multiple techniques including Western blotting, immunohistochemistry, immunocytochemistry, immunofluorescence, and ELISA against known positive and negative samples to ensure specificity and high affinity . For more rigorous validation, researchers might consider using RNA interference (siRNA or shRNA) to knockdown CBR1 expression and confirm corresponding reduction in antibody signal, or alternatively, overexpression systems to demonstrate increased signal intensity . Additionally, researchers working with complex tissue samples should consider performing mass spectrometry analysis of immunoprecipitated proteins to confirm the identity of the proteins recognized by the antibody, especially in cases where potential cross-reactivity with related carbonyl reductases might occur .

What are the optimal conditions for using CBR1 monoclonal antibodies in Western blotting experiments?

Optimizing Western blotting conditions for CBR1 monoclonal antibodies requires careful consideration of several technical parameters to ensure specific and robust detection. Researchers should begin by preparing samples with appropriate lysis buffers containing protease inhibitors, followed by determination of protein concentration to ensure consistent loading across all lanes . For CBR1 detection, typical working dilutions for primary antibodies range from 1:1000 to 1:2000, though this may vary depending on the specific antibody and the abundance of CBR1 in the samples . The calculated molecular weight of CBR1 is approximately 30.4 kDa, providing a reference point for identifying the correct band on immunoblots . Blocking solutions containing 3-5% non-fat dry milk or BSA in TBST are typically effective at reducing background, with incubation times of 1 hour at room temperature or overnight at 4°C for the primary antibody . Secondary antibody selection should match the host species of the primary antibody (typically mouse or rabbit for CBR1 monoclonal antibodies), with HRP-conjugated antibodies being most common for chemiluminescent detection systems . Researchers may need to empirically optimize membrane washing steps, typically using TBST (TBS with 0.05-0.1% Tween-20) with 3-5 washes of 5-10 minutes each to reduce background while preserving specific signals .

How should researchers approach epitope mapping and antibody selection for CBR1 detection in different experimental contexts?

Strategic epitope mapping and antibody selection are crucial for successful CBR1 detection across diverse experimental contexts. Researchers should first consider the specific region of CBR1 recognized by different monoclonal antibodies, as this can significantly impact detection efficiency in different applications . For instance, antibodies targeting conformational epitopes may perform better in applications where protein structure remains intact (like ELISA or IP) but might be less effective in denaturing conditions such as Western blotting . When investigating post-translational modifications or specific functional domains of CBR1, researchers should select antibodies with epitopes that don't overlap with these regions of interest . The clonality of the antibody also merits consideration - monoclonal antibodies like clone 5F10A4 or EPR9660 offer high specificity for a single epitope, which can be advantageous for consistent results across experiments but may be more sensitive to epitope masking in certain sample preparations . Understanding the immunogen used to generate the antibody provides valuable insight, as antibodies raised against full-length recombinant CBR1 (like Boster's M02825) may recognize different epitopes than those raised against specific peptide sequences . For applications requiring detection of protein-protein interactions or CBR1 in complexes, researchers should verify that the selected antibody's epitope remains accessible when CBR1 is engaged in these interactions .

What considerations are important when using CBR1 monoclonal antibodies for immunocytochemistry and immunohistochemistry?

Using CBR1 monoclonal antibodies for immunocytochemistry (ICC) and immunohistochemistry (IHC) applications demands attention to several critical methodological factors. Optimal fixation methods must be determined empirically, as overfixation can mask epitopes while insufficient fixation may compromise cellular architecture; for CBR1 detection, paraformaldehyde fixation (4%) for 15-20 minutes at room temperature often works well for cultured cells, while formalin-fixed paraffin-embedded tissues typically require antigen retrieval steps . Antigen retrieval methods should be optimized based on the specific antibody and tissue type, with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) commonly used for heat-induced epitope retrieval of CBR1 in FFPE samples . For ICC applications, CBR1 monoclonal antibodies are typically used at dilutions around 1:100, though this concentration should be optimized for each specific antibody and cell type . Permeabilization steps are essential for accessing intracellular CBR1, with 0.1-0.5% Triton X-100 or 0.1% saponin commonly used, keeping in mind that excessive permeabilization may compromise cellular structures and epitope integrity . Blocking with 1-5% normal serum from the same species as the secondary antibody helps reduce nonspecific binding, with attention to endogenous peroxidase quenching (typically using 0.3-3% hydrogen peroxide) when using HRP-based detection systems .

How can researchers approach troubleshooting weak or absent CBR1 signals in Western blotting experiments?

When confronting weak or absent CBR1 signals in Western blotting, researchers should systematically evaluate each experimental component to identify and address the underlying issues. First, verify protein loading using housekeeping protein controls and consider increasing the total protein amount loaded per well, as CBR1 expression can vary significantly across different cell lines and tissues . Examine transfer efficiency by staining membranes with Ponceau S or similar reversible stains after transfer, ensuring proteins have successfully migrated from gel to membrane, and consider optimizing transfer conditions for proteins in the 30 kDa range where CBR1 (30.4 kDa) typically appears . Primary antibody concentration may need adjustment, potentially requiring higher concentrations than the manufacturer's recommended 1:1000 dilution, or extending the primary antibody incubation time to overnight at 4°C to enhance signal sensitivity . The detection system's sensitivity might need improvement, either by switching to more sensitive chemiluminescent substrates for HRP-conjugated secondary antibodies or extending film exposure times when using X-ray film-based detection . Consider the possibility that sample preparation has affected epitope integrity, potentially requiring alternative lysis buffers or the addition of phosphatase and protease inhibitors to preserve CBR1 in its recognizable form . If the antibody has been stored improperly or undergone multiple freeze-thaw cycles, its binding capacity may be compromised, necessitating a new antibody aliquot or potentially a different CBR1 antibody clone .

What experimental controls should be included when using CBR1 monoclonal antibodies in various applications?

Incorporating appropriate experimental controls when using CBR1 monoclonal antibodies is essential for result validation and experimental rigor. Positive control samples from tissues or cell lines known to express CBR1 (such as liver tissue extracts or HepG2 cell lysates) should be included to confirm antibody functionality and establish expected signal patterns . Negative controls using tissues or cells with minimal CBR1 expression, or ideally CBR1 knockout models if available, help establish background levels and confirm signal specificity . Technical controls including primary antibody omission controls (applying only secondary antibody) help identify potential non-specific binding of the secondary antibody, while isotype controls using non-specific antibodies of the same isotype (e.g., mouse IgG2a for antibodies like clone BU5F23) help distinguish between specific binding and Fc receptor-mediated or other non-specific interactions . For quantitative applications, researchers should include standard curves using recombinant CBR1 protein at known concentrations, enabling accurate quantification of endogenous CBR1 levels in experimental samples . When examining post-translational modifications or specific activation states of CBR1, controls with appropriate treatments that induce or inhibit these modifications should be incorporated . For multi-color fluorescence applications, single-color controls are essential to establish spectral compensation and identify potential bleed-through when co-staining with other antibodies .

How should researchers approach epitope accessibility issues when CBR1 detection is compromised?

Addressing epitope accessibility problems requires systematic optimization of sample preparation and detection protocols to unmask CBR1 epitopes while maintaining sample integrity. For formalin-fixed tissues showing poor CBR1 detection, researchers should evaluate different antigen retrieval methods including heat-induced epitope retrieval with citrate buffer (pH 6.0), EDTA buffer (pH 8.0-9.0), or enzymatic retrieval using proteinase K, titrating both the duration and intensity of the retrieval process to optimize signal while preserving tissue architecture . When working with native protein applications such as immunoprecipitation or flow cytometry, milder detergents (0.1% Saponin instead of stronger Triton X-100) may preserve structural epitopes while still allowing antibody access to intracellular CBR1 . Sample preparation methods significantly impact epitope availability, so researchers experiencing detection issues should compare different lysis buffers (RIPA versus NP-40 or digitonin-based buffers) that may preserve specific protein conformations and interactions differently . For Western blotting applications with consistent detection problems, consideration of non-reducing conditions might be warranted if the epitope involves cysteine residues affected by reducing agents, though this approach needs careful optimization as CBR1's structure contains important disulfide bonds . Researchers facing persistent accessibility issues might need to consider alternative CBR1 antibody clones that recognize different epitopes, as certain regions of the protein may be consistently masked in particular experimental systems or sample types .

What considerations are important when using CBR1 monoclonal antibodies for investigating CBR1's role in drug metabolism and resistance?

Investigating CBR1's role in drug metabolism and resistance pathways using monoclonal antibodies requires careful experimental design to capture the complexity of these processes. Researchers should select appropriate cellular models that reflect physiologically relevant CBR1 expression levels and activity, such as cardiomyocytes for anthracycline cardiotoxicity studies or cancer cell lines for investigating drug resistance mechanisms . When studying CBR1's involvement in the metabolism of anthracyclines like doxorubicin and daunorubicin to their cardiotoxic metabolites, researchers should incorporate both antibody-based detection of CBR1 expression and enzymatic activity assays to establish correlations between protein levels and functional outcomes . Experimental designs should include appropriate pharmacological inhibitors of CBR1 as controls, enabling researchers to distinguish between CBR1-specific effects and other metabolic pathways that might influence drug metabolism or resistance . Time-course experiments are essential for capturing the dynamic nature of CBR1 expression and activity in response to drug treatments, with careful consideration of both acute and chronic exposure paradigms . For comprehensive studies, combining antibody-based detection methods (Western blotting, IHC, ICC) with functional analyses such as mass spectrometry-based metabolite profiling provides the most complete picture of CBR1's role in converting parent drugs to active or toxic metabolites . When investigating potential post-translational modifications of CBR1 that might affect its drug-metabolizing activity, researchers should employ phospho-specific or other modification-specific antibodies if available, or combine immunoprecipitation with mass spectrometry analyses to identify relevant modifications .