At2g03610 Antibody

What is At2g03610 and why are antibodies against it important in research?

At2g03610 is a gene identifier that appears to be related to the AGTR2 gene family, which encodes angiotensin II receptor type 2. This protein plays crucial roles in brain development and receptor-mediated signaling pathways . Antibodies against this target are valuable research tools that enable detection and measurement of the protein in various biological samples, allowing researchers to investigate its function and expression patterns in different tissues and experimental conditions .

For experimental applications, researchers typically use these antibodies in several key techniques:

• Immunohistochemistry (most common application)

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Western Blot analysis

• Flow cytometry

• Immunofluorescence

The selection of the appropriate antibody depends on the specific research question, tissue type, and detection method employed in the study design.

What are the most effective methods for validating At2g03610 antibody specificity?

Antibody validation is crucial to ensure experimental reliability. For At2g03610 antibodies, a comprehensive validation approach should include:

• Western blot analysis: Look for a single band at the expected molecular weight (~41.2 kilodaltons for human variants) . Multiple bands may indicate cross-reactivity with other proteins.

• Knockout/knockdown controls: Compare antibody staining between wild-type samples and those where At2g03610 expression has been eliminated or reduced through genetic manipulation.

• Peptide competition assays: Pre-incubate the antibody with a synthetic peptide containing the epitope sequence. This should eliminate specific staining in subsequent applications.

• Orthogonal methods: Compare protein expression results with mRNA expression data to confirm correlation.

• Cross-species reactivity testing: If working with multiple model organisms, verify specificity across species of interest.

A well-validated antibody should demonstrate consistent results across multiple validation methods and experimental conditions.

How do you optimize immunohistochemistry protocols for At2g03610 detection in different tissue types?

Optimization of immunohistochemistry (IHC) protocols for At2g03610 detection requires systematic adjustment of several parameters:

• Fixation method: Typically, 4% paraformaldehyde works well for membrane proteins like AT2/AGTR2, but duration may need adjustment (4-24 hours) depending on tissue thickness .

• Antigen retrieval: For formalin-fixed tissues, heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended. Compare both to determine optimal conditions.

• Blocking conditions: Use 5-10% normal serum from the same species as the secondary antibody, with 0.1-0.3% Triton X-100 for membrane permeabilization.

• Antibody dilution: Test a range of dilutions (typically 1:100 to 1:1000) to identify optimal signal-to-noise ratio.

• Incubation conditions: Compare overnight incubation at 4°C versus 1-2 hours at room temperature.

• Detection system: For low abundance targets, consider signal amplification methods like tyramide signal amplification.

For tissues with high autofluorescence or background, include additional blocking steps with avidin/biotin if using biotinylated secondary antibodies, or consider Sudan Black B treatment to reduce autofluorescence.

How can flow cytometry be optimized for detecting At2g03610 expression in heterogeneous cell populations?

Flow cytometry offers powerful capabilities for analyzing At2g03610 expression at the single-cell level. For optimal results in heterogeneous populations:

• Sample preparation: Ensure gentle cell dissociation to preserve membrane integrity, as AT2/AGTR2 is primarily localized to the cell membrane . Use enzymatic methods that minimize epitope damage.

• Fluorochrome selection: Choose fluorochromes with minimal spectral overlap for multiparameter analysis. For AT2 detection alongside other markers, consider bright fluorochromes like PE or APC for lower abundance targets .

• Gating strategy: Implement hierarchical gating including:

  • Viable cell selection (using viability dyes)

  • Doublet discrimination

  • Population-specific markers before analyzing At2g03610 expression

• Controls: Include:

  • Fluorescence minus one (FMO) controls

  • Isotype controls matched to antibody concentration

  • Positive controls (cells known to express At2g03610)

  • Negative controls (cells lacking At2g03610 expression)

• Hybridoma screening approach: For developing new antibodies against At2g03610, fluorescence-activated cell sorting (FACS) can efficiently identify hybridoma cells producing antibodies with high specificity and affinity . This involves labeling the target antigen with fluorescent tags and sorting cells based on fluorescence intensity .

Successful application requires careful titration of antibodies to determine optimal concentration for discrimination between positive and negative populations.

What are the most effective strategies for overcoming cross-reactivity issues with At2g03610 antibodies?

Cross-reactivity presents a significant challenge in antibody-based research. For At2g03610 antibodies, implement these strategies:

• Epitope mapping and selection: Choose antibodies targeting unique epitopes with minimal sequence homology to related proteins. Bioinformatic analysis of epitope sequences prior to antibody selection can identify potential cross-reactivity concerns.

• Pre-absorption techniques: Pre-incubate antibodies with recombinant proteins of potential cross-reactive targets to remove antibodies with undesired binding.

• Competitive binding assays: Develop competition assays using control protein fragments to distinguish specific from non-specific binding. This approach has been successfully used for distinguishing anti-CDR binding from framework binding in therapeutic antibody development .

• Multiple antibody validation: Use multiple antibodies targeting different epitopes of At2g03610 to confirm specificity of observed signals.

• Orthogonal detection methods: Complement antibody-based detection with nucleic acid-based methods (RT-PCR, RNA-seq) to confirm expression patterns.

How do post-translational modifications of At2g03610 affect antibody recognition and experimental interpretation?

Post-translational modifications (PTMs) significantly impact antibody recognition of At2g03610/AGTR2 and can lead to misinterpretation of experimental results:

• Phosphorylation effects: Phosphorylation of serine/threonine residues in AGTR2 can alter antibody epitope accessibility. Site-specific phosphorylation may occur following receptor activation, potentially masking antibody binding sites.

• Glycosylation considerations: As a membrane protein, AGTR2 is subject to N-linked glycosylation which affects protein folding and epitope presentation. Deglycosylation treatments before Western blot analysis may reveal different apparent molecular weights.

• Modification-specific antibodies: Consider using antibodies specifically designed to recognize modified forms of At2g03610 for studying receptor activation states.

• Sample preparation impact: Harsh detergents or reducing conditions may disrupt structural epitopes dependent on disulfide bonds or protein folding.

• Receptor internalization: Activation of AGTR2 can trigger receptor internalization, potentially altering subcellular localization and accessibility to antibodies in intact cells.

To address these challenges, researchers should:

• Use multiple detection methods

• Include appropriate controls for each modification state

• Consider native versus denatured detection methods depending on research question

• Document and report extraction and processing methods in detail to ensure experimental reproducibility

What approaches are most effective for studying At2g03610 interactions with other proteins using antibody-based methods?

Investigating protein-protein interactions involving At2g03610/AGTR2 requires sophisticated antibody-based techniques:

• Co-immunoprecipitation (Co-IP):

  • Use anti-At2g03610 antibodies conjugated to solid supports (e.g., agarose or magnetic beads)

  • Lyse cells under non-denaturing conditions to preserve protein-protein interactions

  • Elute complexes and identify interacting partners by Western blot or mass spectrometry

  • Critical control: Include IgG isotype control immunoprecipitations to identify non-specific binding

• Proximity Ligation Assay (PLA):

  • Allows visualization of protein interactions in situ with single-molecule sensitivity

  • Requires two antibodies from different species targeting At2g03610 and its potential binding partner

  • Each antibody is connected to a different DNA oligonucleotide

  • When proteins are in close proximity (<40 nm), oligonucleotides can be ligated and amplified, creating a fluorescent spot

  • Provides spatial information about interaction locations within cells

• Förster Resonance Energy Transfer (FRET):

  • Label At2g03610 and potential partners with compatible fluorophore pairs

  • Energy transfer between fluorophores occurs only at distances <10 nm

  • Can be combined with antibody-based detection using fluorophore-conjugated secondary antibodies

  • Allows real-time monitoring of dynamic interactions

• Bioluminescence Resonance Energy Transfer (BRET):

  • Similar to FRET but uses luciferase enzyme paired with fluorescent protein

  • Reduces background signal compared to FRET

  • Useful for studying membrane receptor interactions like AGTR2

Each method has distinct advantages for different experimental questions, with Co-IP providing strong evidence of physical association, while PLA and FRET/BRET offer spatial and temporal information about interactions in intact cells.

How can At2g03610 antibodies be effectively used to study receptor signaling pathways?

At2g03610/AGTR2 signaling pathway analysis requires approaches that capture both protein levels and functional states:

• Phospho-specific antibody panels:

  • Use antibodies against phosphorylated downstream signaling molecules

  • Create time-course experiments following receptor stimulation

  • Key targets include MAP kinases, phosphatases, and transcription factors in the AGTR2 pathway

  • Western blot or flow cytometry analysis allows quantification of pathway activation

• Receptor internalization assays:

  • Surface biotinylation followed by antibody detection of internalized receptors

  • Flow cytometry with non-permeabilized cells to quantify surface receptor levels

  • Confocal microscopy with fluorescently-labeled antibodies to track receptor trafficking

• Receptor dimerization analysis:

  • AGTR2 can form homo- and heterodimers affecting signaling properties

  • Use bifunctional cross-linking reagents followed by immunoprecipitation

  • Blue native PAGE with antibody detection to preserve native complexes

• Functional pathway readouts:

  • Combine antibody detection with functional assays (calcium mobilization, cAMP)

  • Correlate receptor levels with signaling outputs

  • Use pathway inhibitors to confirm specificity of observed effects

Research has shown that angiotensin II receptor type 2 (AGTR2/AT2R) mediates protective, anti-inflammatory, and regenerative effects in contrast to the pro-inflammatory AT1R pathway . This highlights the importance of studying differential receptor signaling in disease contexts.

What considerations are important when developing immunoassays for detecting soluble forms of At2g03610?

Development of immunoassays for soluble At2g03610/AGTR2 forms requires attention to several critical factors:

• Epitope accessibility in solution:

  • Membrane proteins like AGTR2 may expose different epitopes when solubilized

  • Test multiple antibody pairs recognizing distinct epitopes

  • Determine if denaturation affects recognition

• Assay format optimization:

  • For sandwich ELISA, evaluate different capture/detection antibody combinations

  • Consider direct coating versus capture antibody approaches

  • Test various blocking reagents to minimize background

• Matrix effects:

  • Biological samples contain interfering substances

  • Develop appropriate dilution protocols for different sample types

  • Include recovery experiments with spiked standards

• Standardization:

  • Use recombinant protein standards with verified concentration

  • Include standard curves in each assay

  • Consider internal controls for inter-assay normalization

• Cross-reactivity testing:

  • Test against related proteins, particularly AGTR1

  • Evaluate species cross-reactivity if developing for multiple research models

  • Perform competition studies with unlabeled antigens

• Preexisting antibodies consideration:

  • Biological samples may contain endogenous antibodies that interfere with detection

  • For therapeutic applications, preexisting anti-hinge or anti-framework antibodies can complicate immunogenicity assessment

  • Develop competition assays that can detect specific anti-CDR antibodies in the presence of preexisting reactivity

These considerations ensure development of robust, specific, and sensitive immunoassays for soluble At2g03610 forms in research and potential clinical applications.

How are single-cell techniques transforming our understanding of At2g03610 expression heterogeneity?

Single-cell technologies are revolutionizing our understanding of At2g03610/AGTR2 expression patterns:

• Single-cell RNA sequencing integration:

  • Correlates At2g03610 transcript levels with protein expression at single-cell resolution

  • Reveals previously unrecognized cellular subpopulations with differential expression

  • Provides context of receptor expression within broader transcriptional programs

  • Challenges: Protein detection requires antibody-based methods like CITE-seq

• Mass cytometry (CyTOF):

  • Uses metal-conjugated antibodies for highly multiplexed protein detection

  • Allows simultaneous measurement of At2g03610 with dozens of other proteins

  • Enables comprehensive phenotyping of expressing cells

  • Metal-tagged antibodies eliminate spectral overlap concerns of fluorescence

• Imaging mass cytometry:

  • Combines CyTOF with imaging to provide spatial information

  • Maps At2g03610 expression within tissue architecture

  • Preserves morphological context lacking in dissociated cell analysis

• Spatial transcriptomics correlation:

  • Links antibody-detected protein localization with spatial gene expression

  • Provides insights into transcriptional regulation in different microenvironments

  • Helps identify factors influencing regional expression differences

These technologies have revealed unexpected heterogeneity in receptor expression between seemingly identical cells and demonstrated differential expression patterns in disease states. For example, in angiotensin receptor research, single-cell approaches have shown that AT2R expression patterns change significantly in response to tissue injury and inflammation .

What novel antibody engineering approaches are improving At2g03610 detection sensitivity and specificity?

Recent advances in antibody engineering are enhancing At2g03610/AGTR2 detection capabilities:

• Recombinant antibody fragmentation:

  • F(ab')2 and Fab fragments reduce background by eliminating Fc-mediated interactions

  • Smaller fragments improve tissue penetration in immunohistochemistry

  • Challenges include potential generation of anti-hinge antibodies that can complicate immunoassay interpretation

• Camelid single-domain antibodies (nanobodies):

  • Exceptionally small size (~15 kDa) enables access to sterically hindered epitopes

  • High stability under various buffer conditions

  • Enhanced penetration into tissue sections

  • Reduced immunogenicity in in vivo applications

• Bispecific antibody formats:

  • Simultaneously bind At2g03610 and a second target

  • Enable novel detection strategies like proximity-based reporter activation

  • Useful for co-localization studies with interaction partners

• Affinity maturation techniques:

  • Directed evolution approaches to enhance binding affinity

  • Phage display with stringent selection conditions

  • Yeast surface display for quantitative screening of binding properties

  • FACS-based hybridoma screening to identify high-affinity antibodies

• Site-specific conjugation:

  • Controlled attachment of detection molecules at defined positions

  • Prevents random labeling that can interfere with antigen binding

  • Engineered cysteine residues or enzymatic tags enable precise modification

These advanced antibody engineering approaches are dramatically improving the toolkit available for At2g03610 research, enabling detection of previously undetectable levels of expression and revealing new biological insights.

How can computational approaches improve At2g03610 antibody selection and experimental design?

Computational methods are increasingly valuable for optimizing At2g03610/AGTR2 antibody research:

• Epitope prediction algorithms:

  • In silico analysis of protein sequence to identify optimal antigenic regions

  • Structural modeling to predict surface-exposed domains

  • Machine learning approaches to identify epitopes with minimal cross-reactivity potential

  • Integration of protein modification data to avoid regions subject to variable PTMs

• Antibody-antigen interaction modeling:

  • Molecular dynamics simulations to predict binding stability

  • Computational docking to estimate binding affinity

  • Identification of critical binding residues to guide mutagenesis studies

• Cross-reactivity prediction tools:

  • Database searching for proteins with similar epitope sequences

  • Proteome-wide analysis to identify potential off-target binding

  • Helps prioritize validation experiments for predicted cross-reactive targets

• Experimental design optimization:

  • Power analysis to determine appropriate sample sizes

  • Batch effect modeling to minimize systematic errors

  • Multiplexed assay design to maximize information from limited samples

• Image analysis automation:

  • Machine learning algorithms for unbiased quantification of immunostaining

  • Deep learning approaches for pattern recognition in complex tissues

  • Standardized analysis pipelines to improve reproducibility

By integrating computational approaches with traditional antibody validation methods, researchers can accelerate discovery while improving reliability. These tools are particularly valuable when working with challenging targets like membrane receptors, where experimental approaches alone may miss important variables affecting antibody performance.

How are At2g03610 antibodies being utilized in understanding disease mechanisms and potential therapeutic targets?

At2g03610/AGTR2 antibodies are providing critical insights into disease processes:

• Cardiovascular disease research:

  • Angiotensin II receptor type 2 (AT2R) has been shown to counteract the harmful effects of AT1R activation

  • Immunohistochemistry with anti-AT2R antibodies reveals expression changes in diseased vessels

  • Helps identify patients who might benefit from targeted therapies affecting the RAS pathway

• Neurological disorders:

  • AGTR2 plays roles in brain development and neural regeneration

  • Antibody-based studies have mapped receptor distribution in different brain regions

  • Expression changes correlate with neurodegenerative conditions

• COVID-19 pathophysiology:

  • The SARS-CoV-2 virus utilizes ACE2 for cell entry, disrupting the renin-angiotensin system

  • Anti-AT1R autoantibodies have been associated with COVID-19 severity

  • Research suggests patients with anti-AT1R autoantibodies may experience protection from severe COVID-19 through altered angiotensin II signaling

  • Increased angiotensin II due to ACE2 occupation by SARS-CoV-2 may become available to bind AT2R, potentially mediating protective effects

• Cancer biology:

  • Altered AGTR2 expression has been detected in various tumors

  • Antibody-based tissue microarray studies correlate expression with patient outcomes

  • Potential target for therapeutic development

• Fibrotic disorders:

  • AT2R activation may counteract pro-fibrotic processes

  • Antibody detection helps quantify receptor levels in fibrotic tissues

  • Expression patterns guide therapeutic strategies targeting the renin-angiotensin system

These applications demonstrate how antibody-based detection of At2g03610/AGTR2 contributes to our understanding of disease mechanisms and identification of potential therapeutic approaches.

What methodological challenges exist when translating At2g03610 antibody-based research findings to clinical applications?

Translating At2g03610/AGTR2 antibody research to clinical settings presents several methodological challenges:

• Standardization across laboratories:

  • Different antibody clones produce variable results

  • Lack of universal calibration standards complicates cross-study comparisons

  • Need for validated reference materials and standardized protocols

• Tissue preservation and processing effects:

  • Clinical samples undergo variable fixation and processing

  • Epitope retrieval methods significantly impact staining patterns

  • Retrospective studies using archived samples face inconsistent preservation

• Quantification challenges:

  • Converting subjective immunohistochemistry scoring to objective measurements

  • Need for digital pathology approaches with validated algorithms

  • Establishing clinically relevant cutoff values for expression levels

• Pre-analytical variables:

  • Time from collection to fixation affects protein preservation

  • Storage conditions impact antigen stability

  • Patient variables (medications, comorbidities) affect expression

• Anti-therapeutic antibody interference:

  • Patients may develop antibodies against therapeutic proteins

  • Preexisting antibodies against structural features (like hinge regions) complicate immunogenicity assessment

  • Requires specialized competition assays to distinguish specific anti-CDR antibodies from framework recognition

• Regulatory considerations:

  • Antibody-based diagnostics require extensive validation

  • Companion diagnostics must demonstrate clinical utility

  • Method transfer between research and clinical laboratories requires rigorous validation

Addressing these challenges requires collaborative efforts between academic researchers, industry partners, and regulatory agencies to establish standardized approaches for translating antibody-based findings to clinical applications.