CYB5R3 Antibody

What is CYB5R3 and what are its primary biological functions?

CYB5R3, also known as NADH-cytochrome b5 reductase 3 or diaphorase-1, is a 301-amino acid protein encoded by the human gene CYB5R3 with a molecular mass of approximately 34 kDa. It catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor . The protein plays crucial roles in multiple cellular processes including the desaturation and elongation of fatty acids, cholesterol biosynthesis, and drug metabolism . In erythrocytes, CYB5R3 is vital for the reduction of methemoglobin, ensuring efficient oxygen transport in the blood . Humans with insufficient CYB5R3 activity suffer from recessive hereditary methemoglobinemia, characterized by severe neurological complications and early childhood death .

What types of CYB5R3 antibodies are available for research and how do they differ?

Various types of CYB5R3 antibodies are available for research applications, including:

• Mouse monoclonal antibodies: Examples include CYB5R3 Antibody (G-11), a mouse monoclonal IgG2a kappa light chain antibody that detects CYB5R3 of mouse, rat, and human origin .

• Rabbit polyclonal antibodies: Such as the rabbit polyclonal CYB5R3 antibody suitable for multiple applications including immunohistochemistry on paraffin-embedded tissues (IHC-P), western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF) .

These antibodies are available in various forms:

• Non-conjugated forms for standard applications

• Conjugated forms including:

  • Agarose conjugates for immunoprecipitation

  • Horseradish peroxidase (HRP) conjugates for enhanced detection

  • Fluorescent conjugates including phycoerythrin (PE), fluorescein isothiocyanate (FITC), and Alexa Fluor® for fluorescence applications

The choice between monoclonal and polyclonal antibodies depends on the specific research application, with monoclonals offering higher specificity but potentially limited epitope recognition compared to polyclonals.

What experimental techniques are most effective for detecting CYB5R3 expression?

Multiple techniques have proven effective for detecting CYB5R3 expression, each with specific advantages:

• Western blotting (WB): Effective for quantifying CYB5R3 protein levels and confirming antibody specificity. Successfully used with cell lysates from various human cell lines including RT4, U-251 MG, A431, and human tissue lysates from liver and tonsil .

• Immunohistochemistry (IHC): Useful for visualizing the spatial distribution of CYB5R3 in tissue sections. Has been successfully performed on formalin-fixed, paraffin-embedded human testis tissue with antibodies at a 1/1000 dilution .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Provides cellular localization information. Successfully performed on PFA-fixed, Triton X-100 permeabilized A431 cells using antibodies at 4 μg/ml concentration .

• Immunoprecipitation (IP): Valuable for protein-protein interaction studies involving CYB5R3 .

• Enzyme-linked immunosorbent assay (ELISA): Useful for quantitative detection of CYB5R3 in solution .

• RNA analysis: Quantitative PCR can be used to measure CYB5R3 mRNA levels, as demonstrated in studies of β-cells from wild type and β-cell-specific FoxO knockout mice .

The selection of technique should be guided by the specific research question and available resources.

How does CYB5R3 function in β-cell physiology and what methodologies best elucidate its role?

CYB5R3 plays a critical role in β-cell function, particularly in mitochondrial function and stimulus/secretion coupling. Research has established that:

• CYB5R3 is a direct target of FoxO1 transcription factor in β-cells, as demonstrated through:

  • Chromatin immunoprecipitation (ChIP) showing FoxO1 enrichment at a putative binding site (5′-ATAAACA-3′, −661 to −667) in the CYB5R3 promoter

  • Adenoviral overexpression of constitutively active FoxO1 (FoxO1-ADA) increasing CYB5R3 expression approximately 5-fold

  • Dominant negative FoxO1 (FoxO1-DN256) suppressing CYB5R3 expression by 60%

• CYB5R3 knockdown significantly impairs β-cell function:

  • Adenoviral shRNA targeting CYB5R3 reduced expression by 95% (mRNA) and 80% (protein)

  • Cells with CYB5R3 knockdown showed impaired glucose-stimulated insulin secretion

  • Basal respiration decreased by approximately 25% in cells stably expressing shCYB5R3

• β-cell-specific CYB5R3 deletion in mice (B-Cyb5r3) revealed:

  • Normal β and δ/pp cell content but increased proportion of α cells (17% vs. 11%, p < 0.05)

  • Approximately 65% decrease in CYB5R3 expression

  • Sex-specific differences in Cre recombination efficiency (less efficient in male mice)

Recommended methodological approaches include:

• Cell sorting techniques to isolate β-cells based on Aldh1A3 activity

• ChIP analysis for transcription factor binding studies

• Adenoviral transduction for gene manipulation

• Seahorse analysis for mitochondrial respiration measurements

• Glucose-stimulated insulin secretion assays

• Immunohistochemistry with careful quantification of islet cell populations

What are the sex-specific differences in CYB5R3 function in cardiovascular tissues and how should experiments be designed to account for these differences?

Research has revealed significant sex-specific differences in the cardiovascular phenotypes resulting from CYB5R3 deletion:

• Male-specific cardiac phenotypes:

  • Male cardiomyocyte-specific CYB5R3 knockout (ac-CYB5R3-KO) mice show a 52% mortality rate within 15 days after tamoxifen treatment

  • These mice exhibit biventricular dilation, significantly increased heart weight-to-body weight ratios, and lung weight-to-body weight ratios

  • Increased LV area and myocyte diameter

  • Elevated WBC, lymphocyte, and monocyte counts

• Female mice show protection from these effects, with no significant cardiac phenotype reported in female ac-CYB5R3-KO mice .

Methodological considerations for studying sex differences in CYB5R3 function:

• Experimental design must include both male and female animals with adequate sample sizes

• Age-matching is critical as hormone levels change throughout life

• When using tamoxifen-inducible systems:

  • Control for potential interactions between tamoxifen and estrogen signaling

  • Use consistent dosing regimens (e.g., 33 mg/kg/day for 5 consecutive days)

  • Time analysis consistently post-induction (e.g., 5 days after treatment)

• Comprehensive phenotyping should include:

  • Survival analysis

  • Cardiac morphometric measurements (heart weight, ventricular dimensions)

  • Histological analysis (myocyte diameter, fibrosis assessment)

  • Complete blood count analysis

  • Cardiac function assessment (echocardiography)

How do genetic variations in CYB5R3 impact protein function and what methodologies can detect functional consequences?

Genetic variations in CYB5R3 have significant implications for protein function and disease susceptibility:

• A serine residue at position 117 of CYB5R3 is predominantly found in individuals of African descent, suggesting potential population-specific variations in enzyme function .

• A variant in the CYB5R3 promoter (43049014 G/T) is associated with fasting glucose levels (P = 2.99 × 10^-4), indicating a potential metabolic role .

• Humans with insufficient CYB5R3 activity develop recessive hereditary methemoglobinemia with severe neurological complications .

Methodological approaches to study genetic variations in CYB5R3:

• Genotyping and sequencing:

  • Targeted sequencing of CYB5R3 coding regions and regulatory elements

  • Genome-wide association studies (GWAS) for metabolic traits

• Functional validation:

  • Site-directed mutagenesis to introduce specific variants

  • CRISPR-Cas9 gene editing to create isogenic cell lines differing only in the variant of interest

  • Enzymatic activity assays measuring:

    • NADH oxidation rates

    • Cytochrome b5 reduction capacity

    • Methemoglobin reduction in erythrocyte models

• Tissue-specific impact assessment:

  • Patient-derived induced pluripotent stem cells (iPSCs) differentiated into relevant cell types

  • Organoid models to study tissue-specific effects

  • Transgenic mouse models expressing human variants

• Population studies:

  • Cohort studies stratified by ancestry

  • Metabolic phenotyping correlated with genetic variations

What are the optimal conditions for Western blot detection of CYB5R3 and how should results be validated?

Optimal Western blot conditions for CYB5R3 detection require careful consideration of several factors:

• Sample preparation:

  • For cell lines: RT4, U-251 MG, A431 cells have shown good CYB5R3 expression

  • For tissues: Human liver and tonsil tissues demonstrate detectable CYB5R3 levels

  • Complete lysis buffers containing protease inhibitors are essential

• Antibody selection and concentration:

  • Anti-CYB5R3 antibody (ab244253) has been successfully used at 0.4 μg/mL concentration

  • Mouse monoclonal IgG2a kappa antibodies like CYB5R3 Antibody (G-11) are effective for human, mouse, and rat samples

• Expected band size and verification:

  • CYB5R3 has a molecular mass of approximately 34 kDa

  • Multiple antibodies should be used to confirm specificity

  • Knockdown or knockout controls are essential for validation

• Validation strategies:

  • Positive controls: Human liver tissue lysate consistently shows strong CYB5R3 expression

  • Negative controls: Include samples from CYB5R3 knockout models where available

  • Peptide competition assays to confirm specificity

  • Comparison of results with different anti-CYB5R3 antibodies targeting distinct epitopes

• Quantification considerations:

  • Normalization to appropriate housekeeping proteins (β-actin, GAPDH)

  • For tissues with variable CYB5R3 expression, consider total protein normalization methods

  • Report results relative to biological controls rather than as absolute values

How should researchers design experiments to investigate CYB5R3's role in mitochondrial function?

Investigating CYB5R3's role in mitochondrial function requires multifaceted experimental approaches:

• Genetic manipulation strategies:

  • RNA interference: shRNA approaches have successfully reduced CYB5R3 expression by 95% (mRNA) and 80% (protein)

  • CRISPR-Cas9 gene editing for complete knockout

  • Conditional deletion systems (e.g., tamoxifen-inducible Cre-loxP) for tissue-specific studies

  • Overexpression systems to assess gain-of-function effects

• Mitochondrial function assessment:

  • Respirometry: Seahorse XF analysis for measuring basal respiration, ATP production, maximal respiration, and spare respiratory capacity

  • CYB5R3 knockdown has been shown to decrease basal respiration by approximately 25%

  • Mitochondrial membrane potential assays using fluorescent probes

  • ROS production assays to assess oxidative stress

  • NAD/NADH ratio measurements

• Tissue-specific considerations:

  • β-cells: Assess glucose-stimulated insulin secretion in conjunction with mitochondrial function

  • Cardiomyocytes: Evaluate contractility and calcium handling alongside mitochondrial parameters

  • Include sex as a biological variable given known sex differences in cardiac phenotypes

• Experimental design table for mitochondrial function studies:

What troubleshooting strategies are recommended when CYB5R3 antibodies show inconsistent results across different samples?

When encountering inconsistent results with CYB5R3 antibodies, consider the following troubleshooting strategies:

• Antibody validation issues:

  • Verify antibody specificity using positive controls (e.g., human liver tissue)

  • Test using CYB5R3 knockout or knockdown samples as negative controls

  • Evaluate cross-reactivity with related proteins like CYB5R4 (knockdown of CYB5R3 has been shown to affect CYB5R4 expression by approximately 30%)

• Sample preparation considerations:

  • CYB5R3 is found in multiple cellular compartments; ensure extraction methods capture relevant fractions

  • For tissue samples, consider perfusion prior to harvest to remove blood contaminants

  • Standardize protein extraction protocols and verify protein integrity

• Technical optimization:

  • Titrate antibody concentrations (successful concentrations range from 0.4 μg/mL for WB to 4 μg/ml for ICC/IF)

  • Optimize incubation times and temperatures

  • For IHC, test different antigen retrieval methods

• Biological variables to consider:

  • Sex differences: Male and female mice show different phenotypes with CYB5R3 deletion

  • Age effects: Expression may vary with developmental stage

  • Cre recombination efficiency: Male mice have shown less efficient Cre-mediated recombination in some models

• Isoform-specific considerations:

  • Confirm which isoform(s) your antibody detects

  • Verify the regional specificity of the antibody (e.g., N-terminal vs. C-terminal epitopes)

  • Consider using antibodies targeting different epitopes to confirm results

What are the most promising research directions for CYB5R3 in metabolic and cardiovascular diseases?

Based on current evidence, several promising research directions for CYB5R3 include:

• Metabolic disease applications:

  • The link between CYB5R3 and glucose homeostasis through β-cell function is particularly significant

  • The association of CYB5R3 promoter variants with fasting glucose levels suggests potential relevance to diabetes pathophysiology

  • Investigation of CYB5R3's role in lipid metabolism could reveal connections to metabolic syndrome

  • Therapeutic approaches targeting CYB5R3 may offer new strategies for preserving β-cell function

• Cardiovascular disease implications:

  • The dramatic cardiac phenotype in male CYB5R3-deficient mice highlights its importance in cardiac health

  • Sex-specific effects suggest hormone-dependent regulation worthy of further investigation

  • CYB5R3's role in mitochondrial function may connect to heart failure pathophysiology

  • Potential therapeutic applications in preventing sudden cardiac death

• Methodological advances needed:

  • Development of small molecule modulators of CYB5R3 activity

  • Tissue-specific delivery systems for therapeutic targeting

  • Advanced imaging techniques to visualize CYB5R3 activity in live cells

  • Multi-omics approaches to understand CYB5R3's role in integrated cellular metabolism