BLVRA Antibody

What is BLVRA and what is its primary biological function?

Biliverdin reductase A (BLVRA), also known as biliverdin-IX α-reductase, is a pleiotropic enzyme that plays a crucial role in the heme catabolic pathway by reducing biliverdin IX α to bilirubin, a potent antioxidant that protects cells from oxidative stress . This enzymatic conversion is vital for maintaining cellular health, as bilirubin has been shown to have anti-inflammatory and cytoprotective properties. BLVRA is primarily expressed in the liver, where it contributes to the detoxification of heme, and its activity is essential for preventing the accumulation of potentially harmful heme breakdown products . The gene encoding BLVRA is located on chromosome 7p14-cen, and it belongs to the GFO/iIDH/MocA family, highlighting its evolutionary significance in metabolic processes .

What types of BLVRA antibodies are available for research purposes?

BLVRA antibodies are available in multiple forms to suit various experimental needs. Researchers can select from both monoclonal and polyclonal antibodies, such as the mouse monoclonal IgG1 kappa light chain antibody (F-1) that detects BLVRA protein in mouse, rat, and human samples . These antibodies come in both non-conjugated forms and various conjugated versions including:

• Agarose-conjugated for immunoprecipitation applications

• Horseradish peroxidase (HRP)-conjugated for enhanced chemiluminescence detection

• Fluorophore-conjugated versions such as phycoerythrin (PE) and fluorescein isothiocyanate (FITC) for flow cytometry and immunofluorescence

• Multiple Alexa Fluor® conjugates for advanced fluorescence imaging applications

The selection of antibody format should be based on the specific experimental technique and detection system being employed in the research protocol.

What are the common applications for BLVRA antibodies in research?

BLVRA antibodies are utilized across multiple experimental techniques in both basic and translational research. The most common applications include:

• Western blotting (WB) for protein expression analysis and quantification

• Immunoprecipitation (IP) to isolate and concentrate BLVRA from complex protein mixtures

• Immunofluorescence (IF) for subcellular localization studies

• Enzyme-linked immunosorbent assay (ELISA) for quantitative measurement of BLVRA levels

• Immunohistochemistry (IHC) for tissue localization and expression pattern analysis

These diverse applications make BLVRA antibodies valuable tools for investigating the enzyme's expression, regulation, and function in various physiological and pathological conditions.

What are the recommended protocols for detecting BLVRA using immunoblotting techniques?

For optimal detection of BLVRA using Western blotting, researchers should consider the following methodological approach:

• Sample preparation: Extract proteins from cells or tissues using RIPA buffer containing protease inhibitors to prevent degradation of BLVRA.

• Protein separation: Load 20-50 μg of total protein onto SDS-PAGE gels (10-12% is optimal for resolving the 33 kDa BLVRA protein) .

• Transfer: Use PVDF membranes for optimal protein binding and signal-to-noise ratio.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute BLVRA antibody as recommended (typically 1/500 - 1/2000 for Western blotting) in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use appropriate species-specific HRP-conjugated secondary antibodies (if using non-conjugated primary antibodies) at 1:5000 dilution.

• Detection: Visualize using ECL substrate and document using a digital imaging system.

The observed molecular weight should be approximately 33 kDa, which corresponds to the calculated molecular weight of BLVRA . When troubleshooting, ensure fresh samples and antibodies are used, as degradation can affect detection sensitivity.

How can researchers optimize immunofluorescence protocols for BLVRA localization studies?

To achieve optimal results in immunofluorescence studies investigating BLVRA subcellular localization:

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature to preserve protein structure while maintaining epitope accessibility.

• Permeabilization: Use 0.1% Triton X-100 in PBS for 10 minutes to allow antibody access to intracellular BLVRA.

• Blocking: Block with 1% BSA and 5% normal serum (from the species of the secondary antibody) for 1 hour to reduce non-specific binding.

• Primary antibody: Dilute BLVRA antibody as recommended (typically 1/100 - 1/200 for immunofluorescence) and incubate overnight at 4°C in a humidified chamber.

• Secondary antibody: Use fluorophore-conjugated secondary antibodies at 1:500 dilution or directly use fluorophore-conjugated BLVRA antibodies such as FITC or PE conjugates .

• Counterstaining: Include DAPI (4',6-diamidino-2-phenylindole) for nuclear visualization, which helps to determine whether BLVRA localizes to the nucleus, cytoplasm, or both compartments .

• Mounting: Use anti-fade mounting medium to preserve fluorescence signal during imaging and storage.

Based on research findings, transduced Tat-BLVRA has been observed in both nuclear and cytosolic compartments , so proper imaging parameters should be set to capture both localizations.

What considerations are important when selecting controls for BLVRA antibody experiments?

Proper experimental controls are critical for ensuring the validity of results when working with BLVRA antibodies:

• Positive controls: Include samples known to express BLVRA, such as liver tissue extracts or hepatocyte cell lines, where BLVRA is abundantly expressed .

• Negative controls: Include samples where BLVRA expression is absent or knockdown models created using RNAi or CRISPR technologies to demonstrate antibody specificity.

• Isotype controls: For flow cytometry or immunostaining applications, include appropriate isotype-matched antibodies (e.g., mouse IgG1 for F-1 clone) to assess non-specific binding .

• Loading controls: For Western blotting, include housekeeping proteins like β-actin, GAPDH, or tubulin to normalize BLVRA expression levels.

• Blocking peptide controls: Use specific BLVRA peptides (matching the immunogen sequence) to pre-absorb the antibody and confirm binding specificity.

• Cross-reactivity assessment: When working across species, validate antibody reactivity in each target species, as antibody performance may vary despite claimed cross-reactivity with human, mouse, and rat BLVRA .

These controls help identify false positive results and ensure that observed signals accurately represent BLVRA expression and localization.

How is BLVRA implicated in cancer progression and what experimental approaches can assess its role?

BLVRA has been implicated in cancer development and progression through several mechanisms that can be investigated using various experimental approaches:

• Expression analysis: BLVRA is overexpressed in multiple cancer types, including colorectal cancer (CRC), where elevated levels correlate with poor prognosis . Researchers can analyze BLVRA expression in clinical samples using ELISA and immunohistochemistry (IHC) techniques.

• Functional studies: To investigate BLVRA's role in cancer:

  • Establish knockdown models using lentiviral vector transfection in cancer cell lines (e.g., HT-29 CRC cells)

  • Create overexpression models (e.g., in SW620 CRC cells) to assess gain-of-function effects

  • Use RT-qPCR and Western blotting to verify altered BLVRA expression levels

• Proliferation assessment: MTT assays can be employed to evaluate how BLVRA expression affects cancer cell proliferation rates. Research has shown that BLVRA overexpression increases proliferation potential in CRC cells .

• Apoptosis analysis: Flow cytometry methods and Western blotting for apoptosis-related proteins can determine how BLVRA influences programmed cell death. Studies indicate that BLVRA overexpression hinders apoptosis in cancer cells .

• Migration and invasion assays: Transwell assays can assess how BLVRA affects cancer cell motility and invasive capacity, with evidence showing enhanced migration and invasion in BLVRA-overexpressing cells .

• EMT marker analysis: Immunofluorescence staining and Western blotting for epithelial-mesenchymal transition (EMT) markers like E-cadherin and vimentin can reveal BLVRA's impact on cancer metastasis. Research demonstrates that BLVRA augments EMT in colorectal cancer .

• Signaling pathway investigation: Western blotting for target proteins in the Wnt/β-catenin pathway can elucidate the molecular mechanisms of BLVRA's oncogenic effects, as this pathway has been implicated in mediating BLVRA's cancer-promoting activities .

These experimental approaches provide comprehensive insights into BLVRA's multifaceted roles in cancer biology and potential as a therapeutic target.

What methodologies can be used to study BLVRA's neuroprotective functions in oxidative stress models?

To investigate BLVRA's neuroprotective properties in oxidative stress conditions, researchers can employ the following methodological approaches:

• Cell viability assays: Utilize neuronal cell lines (e.g., HT-22) exposed to oxidative stressors like hydrogen peroxide (H₂O₂) and assess the protective effects of transduced Tat-BLVRA or other BLVRA expression systems .

• ROS detection: Measure reactive oxygen species using fluorescent probes to quantify how BLVRA modulates oxidative stress levels in neuronal cells under various challenging conditions .

• DNA fragmentation analysis: Assess DNA damage using TUNEL assays or other DNA fragmentation detection methods to evaluate BLVRA's ability to prevent oxidative stress-induced DNA damage .

• Protein transduction strategies: Utilize protein transduction domains (PTDs) like Tat to facilitate BLVRA delivery into cells and tissues for therapeutic applications, enabling assessment of exogenous BLVRA's protective effects .

• Transgenic animal models: Develop BLVRA knockout or overexpression models to study neuroprotection in vivo, particularly in conditions like ischemic brain injury or neurodegenerative disease models .

• Immunohistochemical analysis: Examine BLVRA expression patterns in brain tissues, with particular focus on vulnerable regions like the hippocampal CA1 area in ischemia models .

• Neuroinflammation assessment: Monitor activation of astrocytes and microglia using appropriate markers to determine how BLVRA influences neuroinflammatory responses, which are critical components of neurodegenerative processes .

• Alzheimer's disease model systems: Investigate BLVRA levels in models like triple transgenic AD (3xTg-AD) mice to understand how BLVRA deficiency contributes to increased oxidative stress and tau phosphorylation .

These approaches collectively enable comprehensive investigation of BLVRA's potential as a therapeutic agent for neuroprotection against oxidative stress-related conditions.

How should researchers address discrepancies in BLVRA antibody performance across different experimental systems?

When confronting inconsistencies in BLVRA antibody performance across experimental platforms, researchers should implement a systematic troubleshooting approach:

• Antibody validation: Comprehensively validate each BLVRA antibody using:

  • Multiple detection techniques (WB, IF, IHC, ELISA)

  • Positive and negative control samples with known BLVRA expression profiles

  • Comparison of multiple antibody clones targeting different epitopes

  • Correlation with mRNA expression data from RT-qPCR to confirm protein-level findings

• Species-specific considerations: Despite claims of cross-reactivity with human, mouse, and rat BLVRA , performance may vary significantly between species due to epitope differences. Researchers should:

  • Validate antibodies separately for each species

  • Consider using species-specific antibodies when possible

  • Adjust dilutions and protocols for each species-antibody combination

• Protocol optimization matrix: Develop a systematic optimization matrix varying key parameters:

  • Fixation methods (paraformaldehyde, methanol, acetone)

  • Antigen retrieval approaches (citrate, EDTA, enzymatic)

  • Blocking reagents (BSA, normal serum, commercial blockers)

  • Antibody dilutions and incubation conditions

  • Detection systems (fluorescent vs. chromogenic)

• Context-dependent expression: Consider that BLVRA expression and detectability may be influenced by:

  • Cell/tissue type (liver vs. non-liver tissues)

  • Physiological state (normal vs. stressed conditions)

  • Disease context (cancer vs. non-cancer)

  • Post-translational modifications affecting epitope accessibility

• Batch-to-batch variation: Account for antibody lot variations by:

  • Recording lot numbers and maintaining consistent supplies when possible

  • Re-validating new lots against previous standards

  • Creating internal reference standards for long-term comparability

By systematically addressing these factors, researchers can establish reliable protocols for consistent BLVRA detection across diverse experimental systems.

What are the optimal approaches for quantifying BLVRA expression changes in disease progression studies?

For robust quantification of BLVRA expression changes during disease progression, researchers should employ multi-modal approaches:

• Tissue microarray (TMA) analysis:

  • Develop comprehensive TMAs representing disease progression stages

  • Apply standardized IHC protocols with BLVRA antibodies

  • Implement digital pathology quantification using H-score or Allred scoring systems

  • Correlate BLVRA expression with clinicopathological parameters and patient outcomes

• Multi-omics integration:

  • Correlate protein-level BLVRA measurements with transcriptomic data

  • Perform absolute quantification using:

    • ELISA with recombinant BLVRA standard curves (for tissue/serum samples)

    • Western blotting with densitometric analysis normalized to invariant controls

    • Mass spectrometry-based proteomics for unbiased quantification

• Longitudinal sampling strategies:

  • Collect sequential samples during disease progression

  • Normalize to baseline expression levels within the same patient/model

  • Apply mixed-effects statistical models to account for intra-subject correlation

• Cell type-specific analysis:

  • Employ single-cell approaches to resolve heterogeneous expression patterns

  • Use dual immunofluorescence to correlate BLVRA with cell type markers

  • Apply cell sorting techniques before bulk analysis to enhance resolution

• Functional readouts:

  • Measure BLVRA enzymatic activity alongside protein expression

  • Correlate activity with protein levels to identify post-translational regulation

  • Assess downstream molecular consequences of altered BLVRA expression

A comprehensive scoring system should be developed that integrates multiple parameters:

• Staining intensity (0-3+)

• Percentage of positive cells (0-100%)

• Subcellular localization pattern (nuclear, cytoplasmic, or both)

• Context-specific thresholds defining "overexpression" based on control tissues

This multi-parameter approach provides more meaningful assessment of BLVRA's role in disease progression than single-method quantification strategies.

What experimental design considerations are crucial when investigating BLVRA's dual roles in oxidative stress and signaling pathways?

When designing experiments to elucidate BLVRA's multifunctional roles in both oxidative stress response and signaling pathway modulation, researchers should consider these critical design elements:

• Temporal dynamics integration:

  • Implement time-course experiments capturing both immediate antioxidant effects and delayed signaling responses

  • Design pulse-chase experiments to distinguish between direct enzymatic and secondary signaling functions

  • Use real-time monitoring systems for continuous assessment of ROS levels and signaling pathway activation

• Construct design considerations:

  • Generate separation-of-function mutants that retain enzymatic activity but lack signaling capabilities (or vice versa)

  • Design domain-specific truncation constructs to map functional regions

  • Create fusion proteins with subcellular localization signals to target BLVRA to specific compartments

• Physiologically relevant oxidative stress models:

  • Employ multiple oxidative stressors beyond H₂O₂ (e.g., hypoxia/reoxygenation, glutamate toxicity)

  • Calculate appropriate stressor concentrations that induce stress without overwhelming cellular defense mechanisms

  • Include positive controls with established antioxidants for comparison

• Multi-pathway analysis:

  • Simultaneously monitor Wnt/β-catenin pathway components implicated in BLVRA's cancer-promoting activities

  • Assess potential crosstalk between BLVRA-mediated antioxidant functions and signaling pathway activation

  • Employ pathway inhibitors to dissect interdependencies between oxidative stress responses and signaling cascades

• Combined in vitro and in vivo approaches:

  • Validate cellular findings in animal models of relevant diseases

  • Design conditional knockdown/overexpression systems for tissue-specific and temporal control

  • Develop reporter systems to monitor BLVRA activity in live animals

• Control systems design:

  • Include enzymatically inactive BLVRA mutants to differentiate between catalytic and non-catalytic functions

  • Compare BLVRA with other antioxidant enzymes to establish specificity of observed effects

  • Implement parallel assessment of related family members (e.g., BLVRB) to identify redundant mechanisms

These design considerations enable researchers to untangle BLVRA's complex roles across cellular defense mechanisms and signaling networks, providing deeper insights into its context-dependent functions in health and disease.

How can advanced imaging techniques enhance our understanding of BLVRA's subcellular dynamics?

Cutting-edge imaging approaches offer unprecedented opportunities to visualize BLVRA's dynamic behaviors in living cells:

• Live-cell imaging applications:

  • Generate fluorescent protein-tagged BLVRA constructs (GFP, mCherry) for real-time visualization

  • Employ FRAP (Fluorescence Recovery After Photobleaching) to measure BLVRA mobility between cellular compartments

  • Implement FRET (Förster Resonance Energy Transfer) systems to detect BLVRA interactions with binding partners in situ

• Super-resolution microscopy approaches:

  • Apply STED (Stimulated Emission Depletion) microscopy to resolve BLVRA distribution beyond the diffraction limit

  • Utilize STORM/PALM techniques to achieve nanometer-scale resolution of BLVRA localization

  • Combine with proximity ligation assays to verify protein-protein interactions at molecular resolution

• Correlative light and electron microscopy (CLEM):

  • Identify BLVRA-containing structures by fluorescence then examine ultrastructural context

  • Use immunogold labeling with BLVRA antibodies for high-resolution localization in electron microscopy

  • Map BLVRA distribution relative to cellular membranes and organelles

• Translocation dynamics analysis:

  • Develop biosensor systems to monitor BLVRA translocation between cytosol and nucleus under stress conditions

  • Quantify nuclear/cytoplasmic ratios under various experimental conditions

  • Identify signals that trigger compartmental shifts in BLVRA distribution

• Multi-modal imaging integration:

  • Combine fluorescence microscopy with spectroscopic techniques to simultaneously monitor BLVRA localization and enzymatic activity

  • Implement label-free methods like Raman microscopy to avoid potential artifacts from fluorescent tags

  • Correlate imaging data with biochemical fractionation studies for validation

These advanced imaging approaches provide critical insights into the spatiotemporal regulation of BLVRA function, revealing how its subcellular distribution correlates with its diverse biological roles in normal physiology and disease states.

What methodological approaches are most effective for investigating BLVRA's potential as a therapeutic target?

To thoroughly evaluate BLVRA as a therapeutic target, researchers should implement a comprehensive methodological framework:

• Target validation strategies:

  • Develop conditional knockout models to assess tissue-specific consequences of BLVRA inhibition

  • Create inducible systems to evaluate acute versus chronic BLVRA modulation

  • Use CRISPR-Cas9 screens to identify synthetic lethal interactions with BLVRA in disease contexts

• Small molecule screening approaches:

  • Establish high-throughput enzymatic assays measuring biliverdin to bilirubin conversion

  • Develop cell-based reporter systems to monitor BLVRA activity in intact cells

  • Implement counterscreens to distinguish between enzymatic inhibition and effects on protein-protein interactions

• Protein therapeutics development:

  • Optimize Tat-BLVRA fusion proteins for enhanced cellular uptake and stability

  • Evaluate biodistribution and pharmacokinetics of protein therapeutics in animal models

  • Assess immune responses to recombinant BLVRA therapeutics in preclinical models

• Context-dependent targeting approaches:

  • For cancer applications: Develop strategies to inhibit BLVRA in malignant cells

  • For neuroprotection: Enhance BLVRA delivery or activity in neuronal populations

  • Design tissue-specific delivery systems for targeted BLVRA modulation

• Biomarker development:

  • Identify accessible biomarkers reflecting BLVRA activity (e.g., bilirubin/biliverdin ratios in fluids)

  • Develop companion diagnostics to identify patients likely to respond to BLVRA-targeted therapies

  • Establish pharmacodynamic markers to confirm target engagement in vivo

• Combination therapy evaluation:

  • Test BLVRA modulators with standard-of-care treatments in relevant disease models

  • Identify synergistic drug combinations through matrix screening approaches

  • Evaluate potential for BLVRA status as a predictive biomarker for other therapeutic modalities

These methodological approaches provide a systematic framework for assessing BLVRA's therapeutic potential across multiple disease contexts, from initial target validation through preclinical development of BLVRA-targeting agents.