CBR3 Antibody

What is CBR3 and what is its biological significance?

CBR3 (Carbonyl reductase [NADPH] 3) is an enzyme that catalyzes the NADPH-dependent reduction of carbonyl compounds to their corresponding alcohols. It belongs to the short-chain dehydrogenases/reductases (SDR) family and is expressed in various tissues including liver, kidney, and heart. CBR3 plays a significant role in xenobiotic metabolism and detoxification processes by reducing carbonyl-containing compounds . Its enzymatic activity involves action on several orthoquinones and non-quinone compounds such as isatin and the anticancer drug oracin . The protein has a molecular weight of approximately 30.85 kDa and is also known as SDR21C2 or Short chain dehydrogenase/reductase family 21C member 2 .

What types of CBR3 antibodies are currently available for research?

Multiple types of CBR3 antibodies are available for research purposes, primarily polyclonal antibodies derived from rabbit hosts. Notable examples include polyclonal antibodies that react with human samples (ab196817) suitable for Western blot (WB) and immunohistochemistry on paraffin-embedded sections (IHC-P) , as well as antibodies reactive with human, mouse, and rat samples (A06200) validated for enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and Western blot (WB) applications . Additionally, affinity-purified polyclonal antibodies in IgG format are available for applications such as EIA/RIA, with immunogens typically consisting of full-length or specific amino acid sequences of human CBR3 .

How do I select the appropriate CBR3 antibody for my research?

When selecting a CBR3 antibody, consider these methodological criteria:

• Target species compatibility: Verify that the antibody is reactive to your species of interest. Available antibodies include those reactive to human samples only or those with cross-reactivity to human, mouse, and rat samples .

• Application compatibility: Different antibodies are validated for specific applications. For instance, if performing Western blot analysis, choose an antibody validated for WB with appropriate dilution ranges (typically 1:500-1:2000) .

• Epitope recognition: Consider the immunogen used to generate the antibody. Some are raised against specific peptide regions (e.g., amino acids 151-200 of human CBR3) , while others may target the full-length protein (amino acids 1-277) .

• Validation data: Review the manufacturer's validation images and data to ensure the antibody demonstrates specificity and sensitivity in applications similar to your experimental design.

• Storage and handling requirements: Factor in practical considerations such as storage conditions (-20°C for long-term, 4°C for short-term storage) and avoid repeated freeze-thaw cycles .

What are the optimal dilution ranges for CBR3 antibodies in common applications?

The optimal dilution ranges for CBR3 antibodies vary by application and specific antibody:

When optimizing, perform a dilution series experiment to determine the concentration that provides the best signal-to-noise ratio for your specific sample type and detection system. Always include appropriate positive and negative controls to validate specificity .

How should I store and handle CBR3 antibodies to maintain their activity?

For optimal preservation of CBR3 antibody activity:

• Long-term storage: Store at -20°C in aliquots to minimize freeze-thaw cycles . Most CBR3 antibodies are supplied in buffer containing 50% glycerol which prevents freezing damage.

• Working storage: For frequent use over short periods (up to one month), store at 4°C .

• Formulation: Most commercially available CBR3 antibodies are supplied in PBS containing 50% glycerol, 0.5% BSA (or similar protein stabilizer), and 0.02% sodium azide as preservative .

• Thawing: Thaw frozen antibodies completely at room temperature or 4°C before use. Avoid rapid temperature changes that can denature proteins.

• Handling: After use, return to appropriate storage temperature promptly. Keep track of freeze-thaw cycles and consider creating working aliquots from stock solutions.

What controls should I include when using CBR3 antibodies in my experiments?

Rigorous experimental design requires appropriate controls:

• Positive tissue/cell controls: Include samples known to express CBR3, such as liver, kidney, or heart tissue, which are documented to express the protein .

• Negative controls:

  • Antibody omission control (primary or secondary antibody replaced with buffer)

  • Isotype control (non-specific IgG from the same host species)

  • Tissue negative control (tissues known not to express CBR3)

• Blocking peptide control: If available, pre-incubate the antibody with the immunogenic peptide used to generate it, which should abolish specific staining .

• Knockdown/knockout validation: For definitive specificity confirmation, compare staining between wild-type samples and those with CBR3 gene expression reduced or eliminated.

• Loading controls: For Western blot, include housekeeping proteins (β-actin, GAPDH) to normalize protein loading across samples.

How can I validate CBR3 antibody specificity for my experimental system?

Comprehensive validation strategies for CBR3 antibodies include:

• Multi-technique validation: Confirm CBR3 expression using complementary techniques such as Western blotting, immunohistochemistry, and mass spectrometry.

• Molecular weight verification: CBR3 has a calculated molecular weight of 30.85 kDa . Verify that your antibody detects a protein of appropriate size on Western blots.

• Genetic manipulation approaches:

  • siRNA/shRNA knockdown of CBR3 should reduce antibody signal

  • CRISPR/Cas9 knockout should eliminate specific signal

  • Overexpression of tagged CBR3 should produce increased and co-localizing signal

• Cross-reactivity assessment: Test the antibody against related proteins, particularly CBR1 and CBR4, to ensure specificity within the carbonyl reductase family.

• Epitope mapping: If working with multiple anti-CBR3 antibodies, use those targeting different epitopes to confirm consistent detection patterns.

• Preabsorption testing: Compare staining with antibody alone versus antibody preincubated with immunogenic peptide to confirm signal specificity .

What methodological approaches can resolve inconsistent results with CBR3 antibodies?

When encountering inconsistent results with CBR3 antibodies:

• Sample preparation optimization:

  • For protein extraction, test different lysis buffers containing appropriate protease inhibitors

  • For fixed tissues, assess different fixation protocols (duration, fixative type) as overfixation can mask epitopes

  • For frozen sections, optimize section thickness and fixation method

• Epitope retrieval enhancement:

  • Test different antigen retrieval methods (heat-induced versus enzymatic)

  • Optimize pH and buffer composition for heat-induced epitope retrieval

  • Extend retrieval time for difficult samples

• Signal amplification:

  • Implement tyramide signal amplification for low-abundance detection

  • Use polymer-based detection systems for enhanced sensitivity

  • Consider biotin-streptavidin systems while monitoring endogenous biotin

• Antibody cocktail approach: Combine multiple validated CBR3 antibodies targeting different epitopes to increase detection probability.

• Buffer optimization: Adjust blocking reagents, antibody diluents, and washing buffer compositions to reduce background and enhance specific signal.

• Capture variables: Systematically document all experimental variables (reagent lots, incubation times, temperatures) to identify potential sources of inconsistency.

How should I approach species cross-reactivity when working with CBR3 antibodies?

When working across species with CBR3 antibodies:

• Sequence homology analysis: Analyze the sequence conservation of CBR3 between your species of interest and the immunogen species. CBR3 shows variable conservation across species, which affects antibody cross-reactivity.

• Epitope-specific considerations: Determine if the antibody's epitope (e.g., amino acids 151-200 in human CBR3 ) is conserved in your target species by performing sequence alignments.

• Validation in each species: Even if an antibody is reported to cross-react with multiple species (human, mouse, rat) , independently validate specificity in each species through:

  • Western blot to confirm correct molecular weight

  • Positive and negative tissue controls specific to each species

  • Genetic manipulation controls when available

• Dilution optimization: Optimal antibody dilutions often differ between species; perform species-specific titration experiments.

• Detection system adaptation: Secondary antibody selection and detection systems may need species-specific optimization, particularly for tissues with high endogenous peroxidase or biotin.

How can I optimize CBR3 detection in different subcellular compartments?

CBR3 can be found in different subcellular locations depending on the cell type and physiological conditions. To optimize detection:

• Subcellular fractionation: Perform differential centrifugation to isolate cytosolic, mitochondrial, nuclear, and membrane fractions before Western blot analysis to determine compartment-specific expression.

• Immunofluorescence co-localization: Co-stain with established markers for subcellular compartments:

  • Cytosol: β-tubulin or GAPDH

  • Mitochondria: TOMM20 or Cytochrome c

  • Endoplasmic reticulum: Calnexin or PDI

  • Nucleus: DAPI or Lamin B1

• Fixation optimization: Different fixatives preserve subcellular structures differently:

  • Paraformaldehyde (4%): Good for general structure preservation

  • Methanol/acetone: Better for certain epitopes but disrupts membrane structures

  • Glutaraldehyde: Superior ultrastructure preservation but may reduce antibody accessibility

• Super-resolution microscopy: Techniques such as STED, PALM, or STORM provide improved resolution for precise subcellular localization beyond the diffraction limit.

• Electron microscopy immunogold labeling: For definitive ultrastructural localization, optimize immunogold protocols with different gold particle sizes for potential co-localization studies.

What experimental approaches can correlate CBR3 expression with enzymatic activity?

To establish relationships between CBR3 protein expression and its functional activity:

• Combined immunoblotting and activity assays: Measure CBR3 protein levels by Western blot alongside enzymatic activity using substrates like 1,2-naphthoquinone (reported as CBR3's best substrate ) in the same samples.

• NADPH consumption assays: Monitor NADPH oxidation spectrophotometrically at 340 nm in the presence of various carbonyl substrates, correlating consumption rates with CBR3 expression levels.

• Substrate-specific activity profiling: Test activity against multiple substrates including isatin and orthoquinones, as CBR3 has different catalytic efficiencies for various compounds .

• Genetic manipulation approaches:

  • Overexpression: Transfect cells with CBR3 expression constructs and measure corresponding increases in enzymatic activity

  • siRNA knockdown: Correlate reduced CBR3 protein levels with diminished enzymatic function

  • Site-directed mutagenesis: Create catalytically inactive mutants to serve as negative controls

• In situ activity assays: Develop histochemical approaches to visualize CBR3 activity directly in tissue sections, potentially using electron-accepting dyes that change color upon reduction.

What are the most effective strategies for studying CBR3's role in xenobiotic metabolism?

To investigate CBR3's function in xenobiotic metabolism and detoxification:

• Pharmaceutical substrate panel testing: Assess CBR3's activity against clinically relevant drugs known to undergo carbonyl reduction, such as the anticancer drug oracin .

• Metabolite identification: Use liquid chromatography-mass spectrometry (LC-MS/MS) to identify and quantify specific metabolites produced by CBR3-mediated reduction.

• Cell-based toxicity models: Compare toxicity of carbonyl-containing compounds in:

  • Cells with normal CBR3 expression

  • CBR3-overexpressing cells

  • CBR3-knockdown/knockout cells

• Tissue-specific expression correlation: Quantify CBR3 expression across tissues (liver, kidney, heart) and correlate with tissue-specific metabolic capacity for carbonyl-containing xenobiotics.

• Inhibitor studies: Use selective inhibitors to distinguish CBR3 activity from other carbonyl-reducing enzymes (particularly CBR1) when studying xenobiotic metabolism.

• Polymorphism impact assessment: Investigate how natural genetic variations in the CBR3 gene affect enzyme function and xenobiotic metabolism using recombinant protein variants.

How can I design experiments to distinguish CBR3 function from other carbonyl reductases?

Differentiating CBR3 activity from related enzymes requires specialized approaches:

• Substrate selectivity profiling: CBR3 shows distinct substrate preferences compared to CBR1, with higher activity toward orthoquinones but not paraquinones . Design experiments using discriminating substrates like 1,2-naphthoquinone (preferred by CBR3).

• Selective inhibition strategies: Develop or utilize inhibitors with differential effects on CBR family members to parse their individual contributions in complex systems.

• Expression pattern analysis: Compare tissue and cellular expression patterns of CBR family members using validated antibodies specific to each isoform.

• Kinetic parameter determination: Measure and compare KM and kcat values for CBR3 versus other reductases across substrate panels to establish functional fingerprints.

• Structural biology approaches: Utilize X-ray crystallography or cryo-EM structures (if available) to identify unique binding pocket features that could inform selective substrate or inhibitor design.

• Gene editing combined with rescue experiments: Create CBR3 knockout systems, then selectively reintroduce CBR3 or other family members to isolate their functional contributions.

What techniques are most effective for studying post-translational modifications of CBR3?

To investigate post-translational modifications (PTMs) affecting CBR3 function:

• Phosphorylation analysis:

  • Phospho-specific antibodies if available

  • Phos-tag SDS-PAGE to separate phosphorylated forms

  • Mass spectrometry with phosphopeptide enrichment

• Ubiquitination and SUMOylation detection:

  • Immunoprecipitation under denaturing conditions followed by ubiquitin/SUMO blotting

  • Tandem ubiquitin binding entity (TUBE) pulldown for polyubiquitinated forms

  • Mass spectrometry with GG-remnant-specific enrichment

• Glycosylation assessment:

  • Lectin blotting or affinity purification

  • PNGase F or Endo H treatment to remove N-linked glycans

  • Mass shift analysis by Western blot

• PTM site mapping:

  • Site-directed mutagenesis of predicted modification sites

  • Functional consequences of PTM site mutations

  • Bioinformatic prediction validated by experimental confirmation

• PTM dynamics in response to stimuli:

  • Time-course experiments following exposure to oxidative stress

  • Inhibitor studies targeting specific PTM-regulating enzymes

  • Correlation of PTM status with enzymatic activity

How can advanced computational methods enhance CBR3 antibody-based research?

Integrating computational approaches with antibody-based CBR3 research:

• Epitope prediction and antibody design: Computational tools can identify optimal epitopes for antibody generation with improved specificity, particularly for distinguishing between CBR family members .

• Structure-based epitope mapping: If CBR3 structural data is available, computational mapping of antibody recognition sites can inform experimental design and interpretation.

• High-throughput sequence analysis: Leverage next-generation sequencing to analyze antibody selection experiments, identifying sequence-function relationships for improved CBR3 binding .

• Machine learning for specificity prediction: Train algorithms on existing antibody binding data to predict cross-reactivity and optimize specificity profiles .

• Molecular dynamics simulations: Model antibody-CBR3 interactions at atomic resolution to understand binding mechanisms and predict effects of experimental conditions.

• Systems biology integration: Combine antibody-based CBR3 quantification with multi-omics data to place CBR3 in broader biological networks and predict functional relationships.

What are the most common technical challenges when working with CBR3 antibodies and how can they be addressed?

Frequent challenges and their solutions include:

• High background in immunohistochemistry/immunofluorescence:

  • Increase blocking time and concentration (5% BSA or 10% normal serum)

  • Optimize antibody concentration through titration experiments

  • Include 0.1-0.3% Triton X-100 in blocking and antibody solutions

  • Increase washing duration and buffer volume

  • Use fluorophore-conjugated secondary antibodies with minimal spectral overlap

• Multiple bands in Western blot:

  • Verify sample preparation (complete denaturation, fresh protease inhibitors)

  • Increase membrane blocking time and optimize primary antibody dilution

  • Test different detergents in lysis buffer (RIPA vs. NP-40)

  • Consider native vs. denatured protein conformation effects on epitope accessibility

  • Validate with genetic knockdown to identify specific band

• Poor reproducibility between experiments:

  • Standardize protocols with detailed documentation of all parameters

  • Use consistent antibody lots when possible

  • Prepare master mixes of reagents to minimize pipetting variation

  • Include internal reference standards on each blot/slide

  • Control environmental factors (temperature, humidity) during critical steps

• Low signal strength:

  • Optimize protein loading (for Western blot) or cell/tissue fixation methods

  • Extend primary antibody incubation time (overnight at 4°C)

  • Implement signal amplification systems (HRP polymers, tyramide amplification)

  • Reduce washing stringency while maintaining specificity

  • Evaluate epitope retrieval methods for masked epitopes

How should I approach validation of novel CBR3 antibodies for specific research applications?

When validating new CBR3 antibodies:

• Multi-platform verification: Test across complementary techniques (Western blot, immunoprecipitation, IHC, flow cytometry) to establish application-specific performance.

• Genetic controls: Use CRISPR/Cas9 knockout or siRNA knockdown systems to confirm specificity, comparing signal between normal and CBR3-depleted samples.

• Recombinant protein controls: Test antibody against purified recombinant CBR3 and related family members (CBR1, CBR4) to assess cross-reactivity.

• Epitope mapping: Determine the specific binding region using truncated protein constructs or peptide arrays, especially important for distinguishing between CBR family members.

• Application-specific optimization:

  • For IHC: Test multiple fixation and antigen retrieval methods

  • For IP: Optimize lysis conditions and antibody-to-protein ratios

  • For flow cytometry: Determine optimal permeabilization method for intracellular staining

• Comparison with established antibodies: Benchmark performance against previously validated CBR3 antibodies when available.

• Reproducibility assessment: Validate across multiple biological replicates and different sample types (cell lines, primary cells, tissues) relevant to your research question.