CBR4 Human

What is the molecular characterization of CBR4 in humans?

CBR4 (carbonyl reductase 4) is a protein with a calculated molecular weight of 25 kDa, though it is typically observed at approximately 28 kDa in experimental applications. The protein is encoded by the CBR4 gene (Gene ID: 84869, GenBank Accession Number: BC033650) and consists of 237 amino acids. Its UniProt ID is Q8N4T8 . The slight discrepancy between calculated and observed molecular weights may be attributed to post-translational modifications, which researchers should consider when analyzing experimental results.

What detection methods are most effective for studying CBR4?

Several validated methodologies exist for CBR4 detection, each with specific applications and recommended protocols:

When designing experiments, researchers should optimize dilutions based on their specific sample types and experimental conditions. The polyclonal antibody 13725-1-AP has demonstrated reactivity with human, mouse, and rat samples, making it versatile for comparative studies across species .

What are the key considerations for CBR4 antibody handling and storage?

For optimal antibody performance when studying CBR4, store antibody solutions at -20°C in PBS containing 0.02% sodium azide and 50% glycerol (pH 7.3). Most antibody preparations remain stable for one year post-shipment when stored properly. For smaller aliquots (20μl), the addition of 0.1% BSA helps maintain stability. Aliquoting is generally unnecessary for -20°C storage, which simplifies handling protocols . When designing experimental timelines, consider that repeated freeze-thaw cycles may compromise antibody performance, potentially affecting experimental reproducibility.

How should controls be incorporated in CBR4 experimental design?

Effective experimental design for CBR4 research requires appropriate controls to ensure validity. For immunodetection methods, include:

• Positive controls: HepG2 or BxPC-3 cell lysates, which have been validated for CBR4 expression

• Negative controls: Samples known to lack CBR4 expression or isotype controls for antibody specificity

• Loading controls: Housekeeping proteins (e.g., β-actin, GAPDH) for Western blot normalization

• Blocking peptide controls: To confirm antibody specificity

Control selection should be guided by quasi-experimental design principles that minimize bias while accommodating practical research constraints . When reporting results, clearly document control selection rationale to enhance experimental reproducibility and facilitate meta-analysis across studies.

How can researchers optimize CBR4 immunohistochemistry in different tissue types?

Optimizing CBR4 immunohistochemistry protocols requires careful consideration of tissue-specific factors. While the standard protocol recommends antigen retrieval with TE buffer (pH 9.0), some tissues may require alternative approaches using citrate buffer (pH 6.0) . A systematic optimization strategy includes:

• Tissue fixation assessment: Different fixation durations may affect epitope availability

• Antigen retrieval comparison: Test both recommended buffers (TE pH 9.0 and citrate pH 6.0)

• Antibody titration: Evaluate a range of dilutions (1:20 to 1:200) for optimal signal-to-noise ratio

• Incubation condition variation: Test temperature (4°C, room temperature) and duration variables

• Detection system comparison: DAB vs. fluorescent detection methods

Researchers should implement a factorial experimental design to systematically evaluate these variables, as this approach can efficiently identify optimal conditions while accounting for potential interactions between variables . Document all optimization steps meticulously to facilitate reproducibility across laboratory settings.

What are the best approaches for investigating CBR4 protein-protein interactions?

Investigating CBR4 protein-protein interactions requires multiple complementary methodologies to generate robust evidence. Based on validated protocols, consider the following experimental workflow:

• Immunoprecipitation: Use 0.5-4.0 μg of CBR4 antibody per 1.0-3.0 mg of protein lysate, preferably from HepG2 cells where IP has been validated

• Confirmation with reverse IP: Pull down with antibodies against suspected interacting partners

• Proximity ligation assay: Visualize interactions in situ within cellular contexts

• Mass spectrometry following IP: Identify novel interaction partners

• Functional validation: siRNA knockdown of interaction partners to assess functional relevance

When designing these experiments, researchers should consider randomized block designs to control for batch effects, particularly for multi-day experimental protocols . This approach helps minimize technical variability that could confound interpretation of biological interactions.

What experimental designs are most appropriate for studying CBR4 in complex disease models?

Studying CBR4 in disease contexts requires careful experimental planning to account for biological complexity and variability. For robust findings, consider implementing:

• Stepped-wedge or wait-list cross-over designs: Particularly valuable for intervention studies in patient cohorts, these quasi-experimental approaches allow all participants to eventually receive interventions while maintaining some elements of randomization

• Factorial designs: When investigating multiple factors affecting CBR4 expression or function (e.g., disease state, genetic background, treatment conditions), factorial designs efficiently evaluate interaction effects with fewer experimental units

• Longitudinal sampling: Particularly important for hepatic studies, given CBR4's detection in hepatocirrhosis tissue

• Power analysis: Conduct proper sample size estimation before experimentation to ensure adequate statistical power, as reporting of this critical step has increased from 5% in 2005 to 17% in 2015 in animal studies

When implementing these designs, maintain rigorous blinding procedures and randomization steps to minimize bias, particularly in disease models where subtle effects may be present .

How can researchers integrate CBR4 findings into broader metabolic pathway analyses?

CBR4, as a carbonyl reductase, likely plays important roles in metabolic pathways. To place CBR4 research in broader context:

• Metabolomics integration: Combine CBR4 expression/activity data with untargeted metabolomics to identify affected metabolic pathways

• Systems biology approaches: Use protein interaction databases with experimentally validated CBR4 interactions to build network models

• Comparative analysis: Leverage CBR4's cross-species reactivity (human, mouse, rat) to understand evolutionary conservation of function

• Tissue-specific pathway modeling: Given CBR4's detection in hepatic tissues , contextualize findings within liver-specific metabolic pathways

When designing these integrative studies, researchers should employ proper controls and randomization techniques throughout all experimental stages to minimize systematic bias . The integration of multiple data types requires careful statistical consideration to avoid spurious correlations.

What are the key considerations for CBR4 knockout or knockdown experimental design?

When designing CBR4 gene manipulation experiments:

• Selection of appropriate model system: Given validated reactivity in human, mouse, and rat systems , researchers should choose models based on research questions and available tools

• Knockout validation strategy: Plan comprehensive validation using:

  • Western blot using validated antibody dilutions (1:500-1:1000)

  • qRT-PCR for transcript quantification

  • Functional assays specific to carbonyl reductase activity

• Control selection: Include appropriate wild-type controls matched for genetic background, age, and environmental conditions

• Phenotypic characterization plan: Design a systematic approach to characterize phenotypes across relevant tissues, particularly focusing on hepatic tissues where CBR4 has been studied

To ensure experimental rigor, implement randomization in treatment assignments and blinding during analysis phases. This has become increasingly standard practice, with blinding procedures reported in 47% of animal studies by 2015, up from 26% in 2005 .

How should researchers design experiments to study CBR4 expression across different pathological conditions?

When investigating CBR4 across pathological states:

• Tissue collection standardization: Implement consistent protocols for tissue acquisition, processing, and storage

• Cohort design considerations:

  • Case-control matching for demographic and clinical variables

  • Longitudinal sampling where feasible

  • Clear inclusion/exclusion criteria documentation

• Detection methodology selection:

  • Western blot for quantitative expression differences using recommended dilutions (1:500-1:1000)

  • IHC for spatial localization using optimized antigen retrieval conditions

  • Consider multiplexing with markers of pathological processes

The experimental design should incorporate elements that control for confounding variables, as recommended in practice-based research settings . Researchers should document all methodological decisions to facilitate reproducibility and potential meta-analysis across studies.

What quality control measures are essential for reliable CBR4 research?

To ensure research reliability:

• Antibody validation: Confirm specificity through:

  • Testing in known positive (HepG2, BxPC-3) and negative control samples

  • Western blot molecular weight confirmation (expected at 28 kDa)

  • Peptide competition assays

• Experimental controls: Include technical replicates (minimum triplicate) and biological replicates (determined by power analysis)

• Data validation approaches:

  • Confirmation with orthogonal methods

  • Independent biological replications

  • Blinded analysis of results

• Standardized reporting: Document:

  • Antibody catalog numbers and lot information

  • Detailed methodological protocols

  • Raw data availability plan

These quality control measures align with the increasing emphasis on experimental rigor, as evidenced by the growing reporting of randomization in experimental studies from 41% in 2005 to 54% in 2015 .

How can researchers effectively analyze conflicting CBR4 data from different experimental approaches?

When facing contradictory results:

• Systematic comparison: Create a comprehensive table mapping methodological differences across studies:

  • Antibody sources and validation methods

  • Sample preparation variations

  • Detection system differences

  • Model system variations

• Hierarchical evaluation: Assess evidence quality based on:

  • Methodological rigor (blinding, randomization, controls)

  • Sample size and statistical power

  • Replication status

  • Technical limitations of each method

• Resolution strategies:

  • Conduct bridging studies that systematically vary individual parameters

  • Employ orthogonal methods for key findings

  • Consider meta-analytical approaches when multiple datasets exist

This systematic approach to reconciling conflicting data aligns with the principles of experimental design in practice-based research settings, where controlling for confounding factors is essential .