BLVRB Antibody

What is BLVRB and why is it important in research?

BLVRB is a key enzyme in hemoglobin metabolism, catalyzing the conversion of biliverdin to bilirubin downstream from heme oxygenase. Unlike BLVRA (Biliverdin Reductase A) which is predominantly expressed in adult tissues, BLVRB is typically expressed during fetal development . The enzyme has gained research significance due to its association with intraplaque hemorrhage (IPH) in atherosclerotic plaques and its role in hematopoietic lineage development. BLVRB functions downstream of heme oxygenase(s)-1 (inducible HMOX1) and -2 (constitutive HMOX2) within the heme degradation pathway . Its study is crucial for understanding various pathological conditions including carotid atherosclerosis and thrombocytosis.

What applications can BLVRB antibodies be used for?

BLVRB antibodies have been validated for multiple research applications including:

• Western blot (recommended dilution: 1:500 - 1:2000)

• Immunohistochemistry on paraffin-embedded tissues (IHC-P, recommended dilution: 1:50 - 1:100)

• ELISA

These applications have been validated using samples from multiple species including human, mouse, and rat tissues . For optimal results, each laboratory should determine ideal dilutions for their specific application.

What is the molecular weight and cellular localization of BLVRB?

BLVRB has a calculated molecular weight of approximately 22kDa, which is consistently observed in Western blot analyses of human, mouse, and rat liver tissues . The protein is primarily localized in the cytoplasm, which is an important consideration when designing experimental protocols for cellular fractionation or immunofluorescence studies .

How should researchers validate BLVRB antibody specificity?

Methodological approach:

• Perform Western blot analysis using positive control samples such as U-87MG or U-251MG cell lines

• Include multiple tissue types (e.g., liver tissue from human, mouse, and rat)

• Run samples under reducing conditions to observe the expected 22kDa band

• Consider using recombinant BLVRB protein as a positive control

• Include a negative control using a non-specific IgG from the same host species

Researchers should observe a specific band at approximately 22kDa under reducing conditions when using validated antibodies such as the Sheep Anti-Human BLVRB Antigen Affinity-purified Polyclonal Antibody .

What are the optimal sample preparation techniques for BLVRB detection?

For plasma BLVRB analysis:

• Collect blood samples in appropriate anticoagulant tubes

• Centrifuge at standardized speeds (typically 1500-2000g for 10-15 minutes)

• Separate plasma and store at -80°C until analysis

• For analysis, use affinity proteomic reagents such as those from the Human Protein Atlas project

• Consider coupling antibodies to color-coded magnetic beads for multiplexed detection

For tissue samples:

• Prepare RIPA-solubilized cytoplasmic lysates for cellular BLVRB functional assays

• For immunohistochemistry, follow standard fixation and embedding protocols

• Use recommended antibody dilutions (1:50 - 1:100 for IHC-P)

How can BLVRB be utilized as a biomarker for intraplaque hemorrhage?

BLVRB has emerged as a potential biomarker for intraplaque hemorrhage (IPH), a hallmark of atherosclerotic plaque instability. Research methodologies to explore this application include:

• Plasma measurements using standardized immunoassays

• Correlation with MRI-detected IPH in carotid plaques

• Longitudinal assessments to monitor for recurrent ischemic events

In a clinical study, plasma BLVRB levels were significantly higher in patients with IPH (737.32 ± 693.21 vs. 520.94 ± 499.43 mean fluorescent intensity, p = 0.033) and in patients who developed recurrent ischemic stroke (1099.34 ± 928.49 vs. 582.07 ± 545.34 MFI) . This suggests BLVRB's potential as both a diagnostic and prognostic marker.

What experimental approaches can detect BLVRB enzymatic activity?

To measure BLVRB enzymatic activity:

• Express recombinant wild-type and mutant BLVRB in bacterial systems

• Perform specific activity determination at 25°C using:

  • Flavin mononucleotide as substrate

  • Pooled biliverdin (BV) dimethyl esters synthesized by coupled oxidation of heme

• For cellular samples, use RIPA-solubilized cytoplasmic lysates as the source

• Monitor the conversion of biliverdin to bilirubin spectrophotometrically

• Consider coupled enzymatic assays to assess NAD(P)H oxidation

How can researchers investigate BLVRB mutations and their functional consequences?

To study BLVRB mutations such as S111L:

• Genotyping approach:

  • Design PCR primers specific to the mutation site (e.g., rs149698066 for S111L)

  • Use appropriate genotyping techniques such as TaqMan assays or sequencing

  • Validate findings in independent cohorts

• Functional characterization:

  • Express wild-type and mutant BLVRB in expression systems

  • Compare enzymatic activities using standardized assays

  • Assess redox coupling and reactive oxygen species (ROS) production

  • Measure substrate/cofactor [α/β dinucleotide NAD(P)H] binding efficiency

The BLVRB S111L mutation has been associated with thrombocytosis and shows functional defects in redox coupling using flavin and biliverdin tetrapyrroles, resulting in exaggerated ROS accumulation .

What are the best approaches for investigating BLVRB expression dynamics during hematopoiesis?

BLVRB shows a striking ~40-fold induction during terminal erythroid formation, suggesting a significant role in hematopoietic development . Researchers can investigate this using:

• Cell-specific expression analysis:

  • Isolate different hematopoietic lineage cells at various developmental stages

  • Perform quantitative PCR using fluorescence-based real-time PCR

  • Use specific primers for BLVRB (primer sequences should be optimized)

  • Compare with other heme degradation pathway genes (HMOX1, HMOX2, BLVRA)

• Functional studies:

  • Perform knockdown/knockout experiments using siRNA or CRISPR-Cas9

  • Assess impact on hematopoietic lineage commitment

  • Evaluate changes in reactive oxygen species and heme metabolites

How can co-localization of BLVRB with other proteins be studied effectively?

To investigate the co-localization of BLVRB with proteins such as HMOX-1 in areas of intraplaque hemorrhage:

• Immunohistochemistry approach:

  • Use sequential tissue sections or dual immunofluorescence staining

  • Apply appropriate antibodies at validated dilutions

  • Include proper controls (isotype and tissue negative controls)

  • Quantify co-localization using digital image analysis software

• Biochemical approach:

  • Perform co-immunoprecipitation studies to assess protein-protein interactions

  • Use proximity ligation assays for in situ detection of protein interactions

  • Consider cell fractionation to determine subcellular co-localization patterns

Evidence shows BLVRB colocalizes with HMOX-1 in areas of IPH in plaques from symptomatic patients, supporting a causal link among plasma BLVRB levels, IPH, and hemoglobin metabolism .

How can BLVRB be used to monitor therapeutic interventions targeting intraplaque hemorrhage?

BLVRB has potential utility in monitoring therapeutic responses to treatments targeting IPH. Methodological approach:

• Establish baseline plasma BLVRB levels before treatment initiation

• Monitor BLVRB levels at predetermined timepoints during treatment

• Correlate changes with clinical outcomes and imaging markers of IPH

• Consider multiplex biomarker panels including BLVRB for comprehensive assessment

In preclinical models, plasma BLVRB levels were assessed following antiangiogenic therapy targeting vascular endothelial growth factor receptor 2 (VEGFR-2) in a mouse vein graft model of IPH, demonstrating its utility in monitoring therapeutic responses .

What are the considerations for establishing reference ranges for BLVRB in different populations?

When establishing reference ranges:

• Account for demographic variations:

  • Age (considering BLVRB's differential expression in fetal vs. adult tissues)

  • Sex (evaluate potential hormonal influences)

  • Ethnicity (assess genetic polymorphism frequencies like S111L)

• Consider disease contexts:

  • Establish separate reference ranges for specific clinical conditions

  • For atherosclerosis studies, consider stratifying by:

    • Presence/absence of IPH on imaging

    • Symptomatic vs. asymptomatic status

    • Plaque characteristics

• Standardize analytical methods:

  • Use consistent antibody clones and detection systems

  • Implement quality control measures including positive and negative controls

  • Consider using recombinant BLVRB as a calibrator for quantitative assays

What are common technical challenges when working with BLVRB antibodies?

Common challenges and solutions:

• Background issues in Western blot:

  • Increase blocking time/concentration

  • Optimize antibody dilution (try 1:1000 as a starting point)

  • Use alternative blocking reagents (BSA vs. milk)

  • Ensure complete washing between steps

• Inconsistent results in IHC-P:

  • Optimize antigen retrieval methods

  • Test different dilutions within the recommended range (1:50-1:100)

  • Ensure proper fixation of samples

  • Consider automated staining platforms for consistency

• Cross-reactivity concerns:

  • Validate antibody specificity using positive and negative controls

  • Consider pre-absorption with recombinant BLVRB

  • Verify results using multiple antibodies targeting different epitopes

How can researchers optimize BLVRB detection in clinical samples?

For optimizing BLVRB detection in clinical samples:

• Sample collection and processing:

  • Standardize collection protocols to minimize pre-analytical variables

  • Process samples within a consistent timeframe

  • Consider protease inhibitors to prevent degradation

• Detection optimization:

  • Establish optimal sample dilutions for different sample types

  • Consider signal amplification methods for low-abundance samples

  • Validate results against established biomarkers or clinical parameters

• Quality control measures:

  • Include internal controls in each assay

  • Implement regular calibration using recombinant standards

  • Consider batch effects when analyzing longitudinal samples