endoub Antibody

What is EDNRB and why are EDNRB antibodies important in research?

EDNRB (Endothelin B Receptor) is a non-specific receptor for endothelin 1, 2, and 3 that mediates its action by association with G proteins, activating phosphatidylinositol-calcium second messenger systems . This receptor is crucial in various biological processes including vascular tone regulation, neural crest development, and melanocyte migration.

EDNRB antibodies enable researchers to detect, quantify, and visualize this receptor in experimental systems, supporting investigations into endothelin signaling pathways implicated in cardiovascular disorders, Hirschsprung's disease, and certain cancers. The availability of specific antibodies against EDNRB facilitates research into receptor localization, expression levels, and functional interactions within cellular contexts.

What are the key specifications to consider when selecting an EDNRB antibody?

When selecting an EDNRB antibody, researchers should carefully evaluate several specifications:

• Host species: Commonly rabbit polyclonal (as seen in products DF7104 and ab117529)

• Applications compatibility: Verify validation for specific techniques (WB, IHC, IF/ICC)

• Species reactivity: Confirmed reactivity (human, mouse, rat) and predicted cross-reactivity with other species

• Immunogen details: Target region (e.g., C-terminus aa 400 to C-terminus for ab117529)

• Molecular weight detection: Approximately 50 kDa (calculated)

• Citation history: Number of publications successfully using the antibody

• Visualization method: Native or conjugated (HRP, fluorescent tags)

The antibody's binding epitope location can significantly impact results, particularly when studying receptor conformational changes or protein-protein interactions. Researchers should select antibodies targeting regions appropriate for their specific research questions.

How do I properly store and handle EDNRB antibodies to maintain optimal activity?

Proper storage and handling of EDNRB antibodies is critical for maintaining their specificity and sensitivity in experimental applications. Follow these methodological guidelines:

• Storage temperature: Store antibodies at -20°C for long-term preservation, with aliquoting to prevent freeze-thaw cycles

• Aliquoting protocol:

  • Use sterile microcentrifuge tubes

  • Prepare 10-20 μL aliquots to minimize freeze-thaw cycles

  • Quick-freeze aliquots in liquid nitrogen before transferring to -20°C

• Working dilution preparation: Dilute only the amount needed for immediate use

• Reconstitution (if lyophilized): Use sterile buffered solutions (PBS with 0.1% BSA)

• Avoiding contamination: Always use clean pipette tips and sterile conditions

• Temperature transitions: Allow antibodies to equilibrate to room temperature before opening to prevent condensation

• Record keeping: Maintain detailed logs of freeze-thaw cycles and usage dates

Implementing these practices can significantly extend antibody shelf-life and preserve optimal binding characteristics, ensuring experimental reproducibility and reliable results.

What are the validated applications for EDNRB antibodies in research settings?

EDNRB antibodies have been validated for multiple research applications, each requiring specific optimization parameters:

For optimal results, researchers should carefully consider:

• Sample preparation methods that preserve epitope accessibility

• Appropriate blocking reagents to minimize background signal

• Incubation conditions (temperature, duration) that maximize specific binding

• Compatible detection systems for desired sensitivity levels

• Validated positive and negative controls for result interpretation

Each application requires specific optimization strategies to achieve reliable and reproducible results .

How do I optimize EDNRB antibody dilutions for different experimental techniques?

Optimizing antibody dilutions is a critical step that directly impacts experimental outcomes. Follow this systematic approach:

• Initial titration matrix:

  • Begin with manufacturer's recommended range

  • Prepare a dilution series (e.g., 1:100, 1:500, 1:1000, 1:2000)

  • Test each dilution against positive and negative controls

• Western blot optimization:

  • Start with 1:1000 dilution in 5% BSA or milk-based blocking buffer

  • Evaluate signal-to-noise ratio at each concentration

  • Adjust secondary antibody concentration proportionally

• Immunohistochemistry optimization:

  • Begin with 1:200 dilution for paraffin sections

  • Assess epitope retrieval methods (heat-induced vs. enzymatic)

  • Optimize incubation time (overnight at 4°C vs. 1-2 hours at room temperature)

• Immunofluorescence refinement:

  • Test lower dilutions (1:50-1:200) due to typically lower sensitivity

  • Evaluate different fixation methods (4% PFA, methanol, acetone)

  • Adjust permeabilization conditions to improve intracellular access

• Quantitative assessment:

  • Calculate signal-to-background ratios for each condition

  • Select the dilution that maximizes specific signal while minimizing background

Systematic optimization ensures reproducible results while conserving valuable antibody resources.

What controls should be included when using EDNRB antibodies in experimental designs?

Rigorous control implementation is essential for valid interpretation of experimental results with EDNRB antibodies:

Essential Controls:

• Positive tissue/cell controls:

  • Placental stem villi vessels (known to express EDNRB)

  • Melanocytes and specific neuronal populations

  • Transfected cell lines overexpressing EDNRB

• Negative controls:

  • Primary antibody omission (technical control)

  • Isotype-matched irrelevant antibody

  • Cultured placental villi smooth muscle cells (low EDNRB expression)

  • Tissues from EDNRB-knockout models

• Absorption/competition controls:

  • Pre-incubation of antibody with immunizing peptide

  • Titration series of blocking peptide to demonstrate specificity

• Antibody validation controls:

  • siRNA or CRISPR knockdown of EDNRB

  • Orthogonal detection methods (qPCR correlation)

  • Multiple antibodies targeting different epitopes

• Loading and processing controls:

  • Housekeeping proteins (β-actin, GAPDH) for western blotting

  • Adjacent section staining with established markers

Incorporating these controls enables confident data interpretation and troubleshooting of experimental variables that might impact antibody performance.

How can I distinguish between EDNRB and related receptors (e.g., EDNRA) in my experiments?

Distinguishing between endothelin receptor subtypes requires careful experimental design and antibody selection:

• Epitope selection strategy:

  • Target unique sequences in the C-terminal domain (aa 400 to C-terminus) where sequence homology is lowest between receptor subtypes

  • Avoid antibodies targeting the highly conserved transmembrane domains

• Cross-reactivity assessment:

  • Perform parallel experiments with EDNRA-specific antibodies

  • Use recombinant proteins of both receptor subtypes in competitive binding assays

  • Validate specificity using cells expressing only one receptor subtype

• Functional discrimination approaches:

  • Combine antibody detection with subtype-specific antagonists (BQ-123 for EDNRA; BQ-788 for EDNRB)

  • Correlate antibody binding with receptor-specific signaling pathways

  • Implement receptor subtype-specific siRNA knockdown validation

• High-resolution techniques:

  • Super-resolution microscopy to visualize distinct receptor populations

  • Proximity ligation assays to detect specific receptor interactions

  • Co-immunoprecipitation with subtype-specific antibodies followed by mass spectrometry

• Bioinformatic analysis:

  • Perform sequence alignment of potential epitopes to identify unique regions

  • Assess antibody recognition sites against the UniProt sequence (P24530 for EDNRB_HUMAN)

Implementing these approaches enables confident discrimination between closely related receptor subtypes in complex biological samples.

What approaches can resolve contradictory results when using different EDNRB antibodies?

When faced with contradictory results using different EDNRB antibodies, employ this systematic troubleshooting approach:

• Epitope mapping analysis:

  • Identify the specific binding regions of each antibody

  • Consider whether post-translational modifications might affect epitope accessibility

  • Evaluate whether different conformational states of EDNRB affect antibody recognition

• Validation through orthogonal techniques:

  • Complement antibody-based detection with mRNA quantification

  • Employ functional assays to correlate with expression data

  • Implement CRISPR/Cas9-mediated tagging of endogenous EDNRB

• Comprehensive antibody validation:

  • Test antibodies in EDNRB-knockout or overexpression systems

  • Perform immunoprecipitation followed by mass spectrometry

  • Evaluate antibody recognition of denatured versus native protein forms

• Statistical analysis approach:

  • Calculate enrichment ratio values from multiple experimental replicates

  • Apply bioinformatic tools for high-throughput analysis and interpretation

  • Implement Bayesian statistical methods to integrate data from multiple antibodies

• Methodological standardization:

  • Normalize sample preparation procedures across experiments

  • Standardize detection methods and quantification parameters

  • Implement blinded analysis to eliminate investigator bias

Through systematic investigation of these factors, researchers can identify the source of discrepancies and develop a reliable experimental framework for EDNRB detection.

How do post-translational modifications affect EDNRB antibody recognition and experimental outcomes?

Post-translational modifications (PTMs) significantly impact EDNRB antibody recognition and must be carefully considered in experimental design:

• Critical PTMs affecting EDNRB recognition:

  • N-glycosylation sites that may shield epitopes

  • Phosphorylation states that alter receptor conformation

  • Ubiquitination that affects receptor trafficking and degradation

• Methodological approaches to address PTM interference:

  • Treatment with specific enzymes (e.g., PNGase F for deglycosylation)

  • Phosphatase treatment to standardize phosphorylation states

  • Use of phospho-specific antibodies to detect specific activation states

• Experimental design considerations:

  • Select antibodies targeting regions less susceptible to PTM influence

  • Implement multiple antibodies recognizing distinct epitopes

  • Compare native versus denatured detection systems

• PTM-specific controls:

  • Include samples treated with PTM-inducing agents

  • Utilize mutant EDNRB constructs lacking specific modification sites

  • Compare receptor behavior in different cellular contexts with varied PTM machinery

• Analytical strategies:

  • Western blot mobility shift assays to detect modified forms

  • Mass spectrometry analysis of immunoprecipitated receptor

  • Combined immuno-affinity purification with PTM-specific detection

Understanding how PTMs affect antibody recognition enables more accurate interpretation of experimental results and development of strategies to distinguish receptor populations with different modification patterns.

How can EDNRB antibodies be utilized in cancer research applications?

EDNRB antibodies offer valuable tools for investigating the role of endothelin signaling in cancer development, progression, and therapeutic response:

• Diagnostic and prognostic applications:

  • Tissue microarray analysis of EDNRB expression across tumor types

  • Correlation of EDNRB levels with clinical outcomes and treatment response

  • Development of image analysis algorithms for quantitative assessment

• Mechanistic investigations:

  • Tracking EDNRB expression during epithelial-mesenchymal transition

  • Monitoring receptor internalization following ligand binding

  • Assessing receptor participation in tumor cell migration and invasion

• Therapeutic development support:

  • Screening for antibodies with receptor-blocking capabilities

  • Validating EDNRB as a target for antibody-drug conjugates

  • Monitoring receptor expression changes following treatment

• Methodological approaches:

  • Multiplex immunofluorescence to assess EDNRB in the tumor microenvironment

  • Chromatin immunoprecipitation to identify transcriptional regulators

  • Flow cytometry and cell sorting of EDNRB-positive populations

• Translational research applications:

  • Patient-derived xenograft models with EDNRB expression profiling

  • Correlation of EDNRB status with circulating tumor cell detection

  • Development of companion diagnostics for EDNRB-targeting therapies

These approaches employ antibodies as both analytical tools and potential therapeutic agents, advancing our understanding of EDNRB's role in cancer biology.

How do EDNRB antibodies contribute to cardiovascular and pulmonary disease research?

EDNRB antibodies provide critical insights into cardiovascular and pulmonary pathophysiology through multiple research applications:

• Vascular biology investigations:

  • Immunolocalization of EDNRB in vessel wall components

  • Assessment of receptor distribution in normal versus pathological vessels

  • Correlation of EDNRB expression with vascular remodeling processes

• Pulmonary hypertension research:

  • Quantification of receptor levels in pulmonary arterial smooth muscle cells

  • Tracking receptor redistribution during hypoxic conditions

  • Evaluation of EDNRB-mediated signaling in therapeutic responses

• Atherosclerosis studies:

  • Detection of EDNRB in atherosclerotic plaques

  • Assessment of receptor expression in inflammatory cell populations

  • Correlation with endothelial dysfunction markers

• Heart failure mechanisms:

  • Analysis of receptor expression in cardiac tissue remodeling

  • Monitoring receptor changes during mechanical stress responses

  • Investigation of EDNRB-mediated fibrosis development

• Experimental models:

  • Immunohistochemical assessment in transgenic models

  • Flow cytometry analysis of circulating cells expressing EDNRB

  • Intravital microscopy with fluorescent antibodies for in vivo tracking

Through these applications, EDNRB antibodies enable researchers to establish mechanistic links between endothelin signaling and cardiovascular pathologies, potentially identifying new therapeutic targets.

How can AI-driven approaches enhance EDNRB antibody design and application?

Recent advances in artificial intelligence are revolutionizing antibody design and application, with particular relevance to EDNRB research:

• AI-driven antibody design platforms:

  • RFdiffusion fine-tuned models can generate human-like antibodies against specific targets

  • AI algorithms capable of designing antibody loops—the flexible regions responsible for binding specificity

  • Computer-generated antibody blueprints unlike any seen during training that can bind user-specified targets

• Implementation methodology:

  • Design of antibodies against EDNRB epitopes that are challenging for traditional approaches

  • Generation of more complete human-like antibodies (single chain variable fragments)

  • Development of antibodies with improved specificity for distinguishing between endothelin receptor subtypes

• Experimental validation requirements:

  • Testing AI-designed antibodies against cellular targets expressing EDNRB

  • Comparison with traditionally developed antibodies for sensitivity and specificity

  • Assessment of performance across multiple application platforms

• Bioinformatic analysis integration:

  • Processing of antibody gene data through specialized protocols

  • Quantification of antibody repertoire and functional mapping

  • Calculation of enrichment ratio values to determine antibody efficacy

• Therapeutic development applications:

  • Design of antibodies that can specifically modulate EDNRB function

  • Creation of antibodies with defined glycoforms for optimal therapeutic properties

  • Development of antibodies that selectively target disease-specific receptor conformations

These AI approaches represent a significant advancement in antibody technology that could accelerate both basic research and therapeutic development targeting EDNRB.

What are the applications of antibody glycoengineering in EDNRB research?

Glycoengineering of antibodies presents innovative opportunities for EDNRB research and therapeutic development:

• Glycoengineering fundamentals:

  • N-glycans on IgG Fc regions directly impact antibody effector functions

  • Enzymes like EndoS and EndoS2 enable specific deglycosylation and remodeling of antibody glycans

  • Glycoengineered antibodies can have precisely defined functional properties

• Methodological approaches:

  • Chemoenzymatic synthesis using glycosynthase mutants of EndoS/EndoS2

  • Transfer of homogenous N-glycans to generate antibodies with defined glycoforms

  • Analysis of glycoform impact on antibody-receptor interactions

• Research applications for EDNRB:

  • Development of antibodies with enhanced binding to EDNRB

  • Creation of diagnostic tools with improved sensitivity and specificity

  • Design of therapeutic antibodies with optimized pharmacokinetic properties

• Functional assessment techniques:

  • Surface plasmon resonance to measure binding kinetics of different glycoforms

  • Cell-based assays to evaluate functional impact on EDNRB signaling

  • In vivo biodistribution studies with differentially glycosylated antibodies

• Therapeutic implications:

  • Enhanced control over antibody effector functions through glycan modification

  • Improved safety and efficacy profiles for therapeutic antibodies

  • Potential for developing antibodies that selectively modulate specific EDNRB functions

Glycoengineering represents a sophisticated approach to fine-tuning antibody properties for both research and therapeutic applications targeting EDNRB.