At2g40925 Antibody

How can I verify the specificity of a commercial AT2 receptor antibody?

Commercial AT2 receptor antibodies often demonstrate poor specificity, with studies showing identical immunoreactive patterns in both wild-type and AT2 receptor knockout samples . To properly verify specificity:

• Use knockout cell lines or tissues as negative controls whenever possible

• Employ multiple antibodies targeting different epitopes of the same protein

• Compare antibody performance across different applications (Western blot, immunoprecipitation)

• Validate with recombinant protein expression systems

Research has demonstrated that knockout cell lines provide superior control compared to other validation methods, particularly for Western blot and immunofluorescence applications .

What applications are most reliable for AT2 receptor antibodies?

Based on experimental evidence, AT2 receptor antibodies show variable reliability across different applications. The antibody ab92445 has been validated for:

• Immunoprecipitation (IP)

• Western blotting (WB)

• Cross-reactivity with mouse, human, and rat samples

How should AT2 receptor antibodies be stored and reconstituted?

For optimal performance of AT2 receptor antibodies:

• Store lyophilized antibodies at -20°C

• After reconstitution, make small aliquots to avoid repeated freeze-thaw cycles

• Spin tubes briefly before opening to prevent loss of material adhering to the cap or tube walls

• Reconstitute only the amount needed for immediate experiments

These storage protocols are critical for maintaining antibody activity and preventing degradation that could compromise experimental results.

How do AT2 receptor antibodies perform in detecting specific signaling pathways?

AT2 receptor antibodies can be valuable tools for studying specialized signaling pathways. The receptor primarily signals via non-canonical G-protein- and beta-arrestin-independent pathways . When using antibodies to investigate these mechanisms:

• Consider that AT2 receptor cooperates with MTUS1 to inhibit ERK2 activation and cell proliferation

• Design experiments that account for pathway-specific protein interactions

• Use multiple antibodies targeting different domains to confirm signaling pathway components

• Include appropriate pathway inhibitors as controls

Understanding these signaling mechanisms is crucial when designing experiments to investigate AT2 receptor function in various physiological and pathological contexts.

What strategies can improve antibody performance in challenging experimental systems?

For improved antibody performance in challenging systems:

• Structure-based stabilization approaches have shown success in enhancing antibody quality and specificity

• For instance, researchers working on malaria transmission-blocking vaccines achieved >25°C higher thermostability compared to wild-type proteins using computational structure-based design

• Similar approaches might benefit AT2 receptor antibody design, potentially improving:

  • Epitope recognition

  • Binding affinity

  • Specificity across applications

These advanced engineering approaches represent the cutting edge of antibody technology that could address longstanding issues with AT2 receptor antibodies.

How can I distinguish between AT1 and AT2 receptor antibody binding in tissues with co-expression?

Distinguishing between these related receptors requires careful experimental design:

• Use competitive binding assays with receptor-specific antagonists

• Perform pre-absorption tests with recombinant AT1 and AT2 receptor proteins

• Include controls with selective receptor knockdown

• Employ dual immunolabeling with validated antibodies targeting each receptor

This differentiation is particularly important given the often opposing physiological effects of AT1 and AT2 receptor activation in many biological systems.

What are the recommended dilutions and sample preparation protocols for AT2 receptor antibody applications?

For Western blot detection, VeriBlot for IP Detection Reagent (HRP) has been used successfully at 1:1000 dilution . Always validate these parameters for your specific experimental system.

How should I design proper controls for AT2 receptor antibody experiments?

A robust experimental design must include these controls:

• Positive controls:

  • Transfected cells overexpressing AT2 receptor

  • Tissues known to express high levels of AT2 receptor (adrenal gland, uterus)

• Negative controls:

  • Knockout cell lines or tissues

  • Isotype control antibody (e.g., Rabbit IgG monoclonal [EPR25A])

  • Peptide competition assays with immunizing peptide

• Additional validation:

  • Parallel testing of multiple antibodies recognizing different epitopes

  • Complementary techniques (RT-PCR, functional assays) to confirm protein expression

Research has shown that an alarming average of ~12 publications per protein target include data from antibodies that fail to recognize the relevant target protein , underscoring the critical importance of proper controls.

What are the best practices for antibody validation in new experimental models?

When introducing AT2 receptor antibodies to new experimental models:

• Begin with comprehensive literature review of receptor expression in your model

• Sequence alignment analysis to assess epitope conservation across species

• Gradual validation approach:

  • Start with Western blot to confirm expected molecular weight

  • Progress to more complex applications (IP, IHC, IF) with appropriate controls

  • Quantify and document batch-to-batch variation

• Consider generating cell lines with tagged AT2 receptor as reference standards

This systematic approach minimizes the risk of false positives and increases confidence in experimental findings.

How can I address multiple bands or unexpected molecular weights in AT2 receptor Western blots?

Multiple bands are a common issue with AT2 receptor antibodies, often indicating:

• Post-translational modifications (glycosylation, phosphorylation)

• Receptor dimerization or complex formation

• Proteolytic degradation during sample preparation

• Non-specific binding

Troubleshooting approaches include:

• Varying lysis buffer composition to preserve protein integrity

• Testing different reducing agents and denaturation conditions

• Membrane fractionation to enrich for receptor-containing fractions

• Peptide competition assays to identify specific vs. non-specific bands

• Comparison with recombinant AT2 receptor protein standards

Research has shown that commercial AT2 receptor antibodies often produce different immunostaining patterns with multiple immunoreactive bands , necessitating careful interpretation.

What approaches can resolve discrepancies between antibody-based and functional results?

When antibody-based detection conflicts with functional data:

• Employ complementary nucleic acid-based techniques (RT-PCR, RNA-seq) to confirm expression

• Use pharmacological approaches with AT2 receptor-specific agonists/antagonists

• Consider receptor internalization, trafficking, or conformational changes that might affect epitope accessibility

• Evaluate potential compensatory mechanisms in your experimental system

• Examine published literature for similar discrepancies and resolution strategies

This integrated approach provides a more complete understanding of receptor biology beyond simple antibody detection.

How can I quantitatively assess antibody quality for AT2 receptor research?

Develop a quantitative assessment protocol:

• Signal-to-noise ratio calculation across multiple experiments

• Titration curve analysis to determine optimal working concentrations

• Cross-reactivity matrix against related proteins (particularly AT1 receptor)

• Reproducibility assessment across different lots

• Comparative ranking against alternative commercial antibodies

The YCharOS initiative provides an excellent framework, having demonstrated that recombinant antibodies generally outperform both monoclonal and polyclonal antibodies across multiple assays .

How might recombinant antibody technology improve AT2 receptor detection?

Recombinant antibody technology offers several advantages:

• Eliminated batch-to-batch variability through defined amino acid sequences

• Potential for structure-guided modifications to improve specificity

• Ability to engineer fragment-based derivatives for specialized applications

• Sustainable production without animal immunization

• Precise epitope targeting for studying receptor domains and conformations

Research has demonstrated that recombinant antibodies outperform both monoclonal and polyclonal antibodies on average across multiple assays , suggesting a promising direction for AT2 receptor research.

What emerging technologies might complement or replace traditional antibody-based AT2 receptor detection?

Several innovative approaches are emerging:

• CRISPR-based tagging of endogenous AT2 receptor

• Aptamer technology as alternative affinity reagents

• Nanobody development for improved tissue penetration

• Proximity labeling methods for studying receptor interactions in situ

• Single-molecule imaging techniques for receptor tracking

These approaches may overcome longstanding limitations of traditional antibody-based detection methods while providing new insights into AT2 receptor biology.