AGTR2 Antibody

What is AGTR2 and why is it a significant research target?

AGTR2 (Angiotensin II Receptor Type 2) is a 7-transmembrane protein belonging to family 1 of G protein-coupled receptors. It binds Angiotensin II and plays a critical role in hemodynamic control. Unlike AGTR1, which promotes vasoconstriction and cell proliferation, AGTR2 functions as a physiological antagonist by inhibiting growth and promoting apoptosis. Additionally, AGTR2 signaling induces nitric oxide release in the heart, kidney, and brain and participates in tissue morphogenesis and repair processes . The canonical human AGTR2 protein consists of 363 amino acid residues with a molecular mass of approximately 41.2 kDa . Its clinical relevance in cardiovascular disorders, developmental processes, and newly discovered connections to cystic fibrosis make it a valuable research target .

What applications are AGTR2 antibodies most commonly used for?

AGTR2 antibodies are utilized across multiple experimental applications, with varying degrees of optimization required for each technique:

• Western Blotting (WB): Most widely used application for detecting AGTR2 protein expression levels and molecular weight verification

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of AGTR2 in solution

• Flow Cytometry: For detecting AGTR2 expression in cell populations, particularly useful for examining expression on cell surfaces

• Immunohistochemistry (IHC): For localizing AGTR2 in tissue sections and determining cellular distribution patterns

Different antibody formats exist for these applications, including unconjugated antibodies for general detection and conjugated formats (such as APC-conjugated) for specialized applications like flow cytometry .

How should researchers validate AGTR2 antibody specificity?

Methodological approach to AGTR2 antibody validation should include:

• Positive and negative control tissues: Validate using tissues known to express AGTR2 at high levels (such as myometrium, fetal kidney, and intestine) and compare with tissues with low or no expression

• Cell line testing: Confirm specificity using established cell lines:

  • Positive samples: HeLa, 293T, PC-12, HepG2, mouse brain, mouse kidney, mouse uterus, and rat liver cells

  • Control comparison: Use isotype control antibodies (e.g., IC0041A for flow cytometry) to assess non-specific binding

• Genetic validation: When possible, compare results between wild-type samples and AGTR2 knockout models to confirm antibody specificity

• Cross-reactivity assessment: Test against related proteins, particularly AGTR1, to ensure target specificity

What are the optimal conditions for using AGTR2 antibodies in Western blotting?

Western blotting with AGTR2 antibodies requires careful optimization:

• Sample preparation:

  • Membrane protein extraction techniques are essential as AGTR2 is a multi-pass membrane protein

  • Avoid excessive heating which may cause aggregation of membrane proteins

  • Include protease inhibitors to prevent degradation

• Dilution optimization:

  • Start with recommended dilution ranges (1:500 - 1:2000 for many AGTR2 antibodies)

  • Perform titration experiments to determine optimal signal-to-noise ratio

• Detection system:

  • Enhanced chemiluminescence systems are typically preferred

  • Consider longer exposure times when detecting native expression levels in tissue samples

• Molecular weight verification:

  • Expect bands at approximately 41 kDa, though post-translational modifications (particularly glycosylation) may alter apparent molecular weight

  • Multiple bands may indicate different glycosylation states or proteolytic processing

How should researchers interpret cross-species reactivity of AGTR2 antibodies?

Cross-species reactivity interpretation requires methodological considerations:

• Sequence homology analysis:

  • Human AGTR2 shares 92% amino acid sequence identity with mouse and rat AGTR2

  • Examine the specific epitope sequence recognized by the antibody

  • Antibodies raised against conserved regions will show broader cross-reactivity

• Empirical validation approach:

  • Always verify cross-reactivity experimentally, regardless of reported species reactivity

  • Use appropriate positive controls from each species

  • Adjust antibody concentration when switching between species (may require higher concentrations for cross-reactive applications)

• Species-specific considerations:

  • Consider species-specific expression patterns (e.g., highly expressed in mouse brain, kidney, and uterus)

  • Be aware that glycosylation patterns may differ between species, affecting antibody recognition

What factors affect AGTR2 antibody selection for flow cytometry applications?

For flow cytometry applications with AGTR2 antibodies, consider these methodological factors:

• Conjugation format:

  • Direct conjugates (e.g., APC-conjugated) eliminate secondary antibody steps and reduce background

  • Fluorophore selection should be compatible with other markers in multi-parameter panels

  • Example: Mouse Anti-Human AGTR2 APC-conjugated Monoclonal Antibody (FAB3659A) used for NS0 mouse myeloma cell line detection

• Clone selection:

  • Epitope accessibility in living cells is critical

  • Clones recognizing extracellular domains are preferred for surface staining

  • Example: Clone #364805 has been validated for flow cytometry of AGTR2

• Protocol optimization:

  • Cell preparation (fixation vs. live staining) affects epitope recognition

  • Buffer composition influences background and specific signal

  • Titration experiments are essential for determining optimal concentration

How does AGTR2 genetic variation influence cystic fibrosis pathophysiology?

Recent research has revealed significant connections between AGTR2 and cystic fibrosis:

• Genetic association evidence:

  • GWAS studies identified variants at the X-chromosome locus containing AGTR2 that significantly associate with lung function in CF patients

  • Quantile regression analysis showed CF patients with the risk genotype (C [males] or CC [females]) had worse pulmonary function compared to those with low-risk genotypes

• Pulmonary function measurements:

  • Statistically significant reductions in multiple spirometric parameters:

• Mechanistic research approaches:

  • AGTR2 deletion or antagonism in CF mouse models shows promising research avenues

  • Utilizing AGTR2 antibodies to monitor receptor expression and localization in CF tissues can provide mechanistic insights

What methodologies are recommended for studying AGTR2-mediated signaling pathways?

Advanced experimental approaches for AGTR2 signaling research:

• Co-immunoprecipitation studies:

  • Use AGTR2 antibodies to pull down receptor complexes

  • Identify interaction partners in different cellular contexts

  • Example: AGTR2 cooperation with MTUS1 to inhibit ERK2 activation and cell proliferation

• Phosphorylation state analysis:

  • Use phospho-specific antibodies to downstream targets

  • Monitor nitric oxide release in response to AGTR2 activation in heart, kidney, and brain tissues

  • Quantify opposing effects to AGTR1-mediated pathways

• Tissue-specific expression profiling:

  • Compare AGTR2 expression between adult tissues (myometrium, adrenal gland, fallopian tube, cerebellum) and fetal tissues (kidney and intestine)

  • Correlate expression levels with signaling pathway activation

How can AGTR2 antibodies be applied in adipose tissue and stem cell research?

Recent studies have revealed novel applications for AGTR2 antibodies in stem cell biology:

• Adipose-derived mesenchymal stem/stromal cells (ADMSCs) research:

  • Flow cytometry with AGTR2 antibodies has demonstrated expression of angiotensin II receptors in ADMSCs

  • Studies show local angiotensin II promotes adipogenic differentiation through AGTR2

• Differentiation monitoring methodology:

  • Use AGTR2 antibodies to track receptor expression changes during differentiation processes

  • Combine with other markers to create comprehensive differentiation profiles

• Comparative receptor analysis:

  • Quantify AGTR1 versus AGTR2 expression ratios in stem cells and during differentiation

  • Correlate receptor balance with cell fate decisions

What are common technical issues when working with AGTR2 antibodies and how can they be resolved?

Methodological approaches to overcome technical challenges:

• High background in immunohistochemistry/immunofluorescence:

  • Increase blocking time and concentration (5% BSA or 10% serum from secondary antibody host species)

  • Use more stringent washing protocols (increase number of washes and duration)

  • Titrate primary antibody to find optimal concentration

  • Try alternative fixation methods that better preserve AGTR2 epitopes

• Poor detection in Western blotting:

  • Optimize protein extraction for membrane proteins (avoid harsh detergents that may denature epitopes)

  • Increase protein loading for tissues with low expression

  • Try alternative transfer methods optimized for hydrophobic membrane proteins

  • Consider native conditions if the antibody recognizes conformational epitopes

• Variable results between experiments:

  • Standardize protein quantification methods

  • Maintain consistent sample preparation protocols

  • Use internal loading controls appropriate for membrane proteins

  • Consider lot-to-lot variation between antibody preparations

How can researchers interpret contradictory results from different AGTR2 antibodies?

When facing contradictory results, implement this systematic approach:

• Epitope mapping analysis:

  • Determine epitopes recognized by different antibodies (N-terminal, C-terminal, transmembrane domains)

  • Antibodies targeting different epitopes may yield different results depending on protein conformation, processing, or interactions

• Validation with orthogonal techniques:

  • Confirm protein expression using mRNA analysis (qPCR, RNA-seq)

  • Employ genetic approaches (siRNA knockdown, CRISPR knockout) to verify specificity

  • Use multiple antibodies targeting different epitopes to build consensus data

• Context-dependent expression consideration:

  • Different experimental conditions may affect AGTR2 expression or accessibility

  • Document and control for variables that might affect receptor expression (cell confluency, passage number, treatments)

What strategies improve detection of low AGTR2 expression levels?

For low abundance AGTR2 detection, employ these methodological improvements:

• Signal amplification techniques:

  • Use tyramide signal amplification for immunohistochemistry

  • Consider biotin-streptavidin systems for enhanced sensitivity

  • Employ more sensitive detection reagents for Western blotting

• Sample enrichment approaches:

  • Use membrane protein extraction and enrichment protocols

  • Concentrate samples before analysis

  • Immunoprecipitate AGTR2 before detection when appropriate

• Alternative detection platforms:

  • Consider more sensitive techniques like multiplexed immunoassays

  • Digital droplet PCR for mRNA expression can complement protein studies

  • Single-cell approaches may reveal heterogeneous expression masked in bulk analysis

How are new technological advances improving AGTR2 antibody applications?

Emerging technologies enhancing AGTR2 research include:

• Super-resolution microscopy applications:

  • Nanoscale localization of AGTR2 in membrane microdomains

  • Co-localization with signaling partners at previously unresolvable resolution

  • 3D reconstruction of receptor distribution in complex tissues

• Multiplex antibody approaches:

  • Simultaneous detection of AGTR2 with multiple signaling partners

  • Spatial transcriptomics combined with protein detection

  • Mass cytometry for high-dimensional analysis of signaling networks

• In vivo imaging applications:

  • Development of antibody fragments for in vivo imaging

  • Near-infrared fluorophore conjugates for deeper tissue penetration

  • PET imaging with radiolabeled antibodies or fragments for whole-organism studies

What are the most promising therapeutic applications of AGTR2 research?

Translational research opportunities emerging from AGTR2 studies:

• Cystic fibrosis interventions:

  • AGTR2 antagonism shows promise in CF models

  • PD123,319 (selective AGTR2 antagonist) demonstrates potential therapeutic benefits in preclinical studies

  • Antibody-based therapeutics targeting AGTR2 represent a future research direction

• Cardiovascular applications:

  • AGTR2 modulation as an alternative approach to traditional angiotensin receptor blockers

  • Tissue-specific targeting to enhance beneficial AGTR2 signaling

  • Precision medicine approaches based on AGTR2 genetic variants

• Developmental disorders:

  • AGTR2's role in tissue morphogenesis suggests potential in developmental disorder treatments

  • Monitoring interventions using antibody-based detection of receptor expression and localization