Phospho-INSR (Tyr1361) Antibody

What is Phospho-INSR (Tyr1361) Antibody and what signaling event does it detect?

Phospho-INSR (Tyr1361) Antibody is a rabbit polyclonal antibody specifically designed to detect the insulin receptor (INSR) only when phosphorylated at tyrosine residue 1361. This antibody recognizes a critical post-translational modification that occurs during insulin signaling cascade activation. The antibody is typically raised against synthetic peptides derived from human insulin receptor around the phosphorylation site of Tyr1361, with an amino acid range of approximately 1331-1380 . The specificity of this antibody allows researchers to distinguish between the active (phosphorylated) and inactive (unphosphorylated) forms of the insulin receptor, providing crucial information about receptor activation status in various experimental conditions .

What experimental applications is Phospho-INSR (Tyr1361) Antibody suitable for?

Phospho-INSR (Tyr1361) Antibody demonstrates versatility across multiple experimental platforms commonly used in molecular and cellular biology research. The antibody is validated for Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and Enzyme-Linked Immunosorbent Assay (ELISA) . These applications enable researchers to investigate insulin receptor phosphorylation in various contexts, from protein expression levels in cell lysates to spatial localization within tissue sections. The recommended dilutions for optimal results vary by application: Western Blot (1/500 - 1/2000), Immunohistochemistry (1/100 - 1/300), and ELISA (1/10000) . Researchers should validate these dilutions in their specific experimental systems for optimal signal-to-noise ratios.

What controls should be included when using Phospho-INSR (Tyr1361) Antibody?

Proper experimental controls are essential for meaningful interpretation of results when using phospho-specific antibodies. Researchers should implement the following controls:

• Loading control: Include a housekeeping protein (e.g., GAPDH, β-actin) to ensure equal protein loading across samples, especially for Western blotting applications .

• Total protein control: Measure the total insulin receptor protein levels (using a non-phospho-specific INSR antibody) alongside the phosphorylated form to distinguish between changes in phosphorylation status versus changes in total protein expression .

• Positive control: Include samples with known INSR Tyr1361 phosphorylation. This can be achieved by treating cells with insulin to stimulate receptor phosphorylation. Typically, insulin stimulation (1 ng/mL for 30 minutes) induces robust phosphorylation at Tyr1361 that can be detected by the antibody .

• Negative control: Include samples where phosphorylation is absent or blocked, such as serum-starved cells or samples treated with specific kinase inhibitors.

How can I validate the specificity of Phospho-INSR (Tyr1361) Antibody?

Validating antibody specificity is crucial for reliable research outcomes. Consider these approaches:

• Peptide competition assay: Pre-incubate the antibody with the phosphorylated peptide used as immunogen. This should abolish specific binding in subsequent assays.

• Phosphatase treatment: Treat half of your positive sample with lambda phosphatase to remove phosphate groups. The signal should disappear in the phosphatase-treated sample if the antibody is truly phospho-specific.

• Knockdown/knockout controls: Use INSR knockdown or knockout models to confirm antibody specificity.

• Insulin stimulation timecourse: Perform a time-dependent stimulation with insulin and monitor Tyr1361 phosphorylation, which should increase after stimulation if the antibody is detecting the correct phosphorylation event.

What is the functional significance of Tyr1361 phosphorylation in insulin signaling?

Tyrosine 1361 phosphorylation represents a critical event in insulin receptor activation and subsequent downstream signaling. When insulin binds to the insulin receptor, it triggers autophosphorylation of several tyrosine residues, including Tyr1361. This phosphorylation site is particularly important for the activation of downstream signaling pathways that mediate the metabolic and mitogenic effects of insulin .

The phosphorylation at Tyr1361 contributes to the activation of two main signaling cascades:

• PI3K-AKT/PKB pathway: This pathway is primarily responsible for the metabolic actions of insulin, including glucose transport via GLUT4 translocation, glycogen synthesis, and inhibition of gluconeogenesis .

• Ras-MAPK pathway: This pathway regulates gene expression and cooperates with the PI3K pathway to control cell growth and differentiation .

Disruption of Tyr1361 phosphorylation can impair insulin signaling, potentially contributing to insulin resistance and related metabolic disorders.

How does Tyr1361 phosphorylation relate to other phosphorylation events in the insulin receptor?

The insulin receptor undergoes multiple phosphorylation events during activation, creating a complex signaling network. Tyr1361 phosphorylation occurs within the context of these multiple phosphorylation events, which collectively determine signaling specificity and intensity. The phosphorylated tyrosine residues serve as docking sites for various signaling proteins containing Src-homology-2 (SH2) domains, including insulin receptor substrates (IRS1-4), SHC, GAB1, and CBL . These interactions initiate diverse downstream signaling cascades that mediate insulin's cellular effects.

How can Phospho-INSR (Tyr1361) Antibody be used to investigate insulin resistance disorders?

Phospho-INSR (Tyr1361) Antibody serves as a valuable tool for investigating the molecular mechanisms underlying insulin resistance disorders. Researchers can apply this antibody in several advanced applications:

• Comparative analysis: Compare Tyr1361 phosphorylation levels between normal and insulin-resistant tissues or cell models to identify signaling defects.

• Drug screening: Evaluate the effect of potential therapeutic compounds on restoring insulin receptor phosphorylation in insulin-resistant models.

• Biomarker development: Explore the potential of Tyr1361 phosphorylation as a biomarker for insulin resistance severity or treatment response.

• Pathophysiological studies: Investigate how specific disease states (obesity, diabetes, etc.) affect insulin receptor phosphorylation patterns.

What is Type B insulin resistance and how does it relate to INSR Tyr1361 phosphorylation?

Type B insulin resistance (TBIR) is an autoimmune disorder characterized by the presence of insulin receptor autoantibodies (InsR-aAb) that interfere with normal insulin signaling. These autoantibodies can directly impair insulin-induced insulin receptor phosphorylation, including at Tyr1361 .

In TBIR research, Phospho-INSR (Tyr1361) Antibody can be used to:

• Evaluate autoantibody effects: Determine whether patient-derived autoantibodies inhibit insulin-induced Tyr1361 phosphorylation in cell culture models.

• Monitor treatment response: Assess the recovery of insulin receptor phosphorylation following immunosuppressive therapy aimed at reducing autoantibody levels.

• Distinguish mechanisms: Differentiate between antagonistic (inhibiting insulin binding) versus post-binding defects in insulin signaling.

Research has demonstrated that immunoglobulins isolated from TBIR patients inhibit insulin-induced phosphorylation of the insulin receptor at Tyr1361, confirming their antagonistic effect on insulin signaling . This inhibition correlates with disease severity and clinical manifestations, highlighting the pathophysiological relevance of this phosphorylation site.

How can Phospho-INSR (Tyr1361) Antibody be used in novel diagnostic assays?

Recent research has developed innovative diagnostic applications using phosphorylation-specific antibodies for the insulin receptor. A novel in vitro assay for detecting insulin receptor autoantibodies in Type B insulin resistance utilizes phospho-specific antibodies (including those targeting pTyr1361) to assess the functional impact of these autoantibodies on insulin signaling .

The assay workflow typically involves:

• Isolating immunoglobulins from patient serum

• Exposing cultured cells (e.g., HepG2 hepatic cells) to these immunoglobulins in the presence or absence of insulin

• Detecting insulin receptor activation using phosphorylation-specific antibodies against pTyr1361

• Quantifying the degree of inhibition compared to control samples

This approach provides both diagnostic information and insights into the biological activity of patient-derived autoantibodies, correlating with clinical disease severity .

What are common technical challenges when using Phospho-INSR (Tyr1361) Antibody?

Researchers working with phospho-specific antibodies often encounter several technical challenges:

• Phosphorylation lability: Phosphorylated proteins are susceptible to dephosphorylation by endogenous phosphatases during sample preparation. Always include phosphatase inhibitors in lysis buffers and maintain samples at cold temperatures.

• Low signal intensity: Phosphorylation is often a transient and substoichiometric modification. Optimize stimulation conditions and consider using phosphatase inhibitors or signal enhancement techniques.

• Non-specific binding: Some phospho-specific antibodies may cross-react with similar phosphorylation motifs. Validate specificity using the controls mentioned earlier.

• Variable phosphorylation kinetics: The timing of Tyr1361 phosphorylation may vary across cell types and experimental conditions. Perform time-course experiments to identify optimal time points for detection.

How should samples be prepared to preserve INSR Tyr1361 phosphorylation?

Preserving phosphorylation status during sample preparation is critical for accurate analysis. Follow these recommendations:

• Rapid sample processing: Minimize the time between cell/tissue collection and protein extraction to prevent dephosphorylation.

• Phosphatase inhibitors: Include a comprehensive phosphatase inhibitor cocktail in all buffers used during sample preparation.

• Cold temperature: Perform all sample preparation steps at 4°C to reduce phosphatase activity.

• Appropriate lysis conditions: Use lysis buffers containing detergents that effectively solubilize membrane proteins like INSR without disrupting phosphorylation.

• Avoid repeated freeze-thaw cycles: Store samples in single-use aliquots to prevent degradation of phosphorylated proteins .

How should Phospho-INSR (Tyr1361) data be quantified and normalized?

Proper quantification and normalization are essential for meaningful interpretation of phosphorylation data:

• Quantification methods:

  • For Western blots: Use densitometry software to quantify band intensity

  • For immunofluorescence: Measure mean fluorescence intensity in defined cellular regions

  • For ELISA: Generate standard curves using recombinant phosphorylated proteins

• Normalization approaches:

  • Normalize phospho-INSR signal to total INSR signal (phospho/total ratio)

  • For Western blots, additionally normalize to loading controls

  • For cell-based assays, normalize to cell number or total protein content

• Statistical analysis:

  • Compare multiple biological replicates (minimum n=3)

  • Apply appropriate statistical tests based on data distribution

  • Report both fold-changes and statistical significance

How can I distinguish between changes in phosphorylation versus changes in expression?

A critical challenge in phosphorylation studies is distinguishing between altered phosphorylation status and changes in total protein expression. To address this:

• Always measure total INSR: Probe parallel samples or strip and reprobe membranes with antibodies against total INSR.

• Calculate phospho/total ratio: Divide the phospho-INSR signal by the total INSR signal to normalize for expression differences.

• Consider additional phosphorylation sites: Examine multiple phosphorylation sites on INSR to determine whether changes are site-specific or affect all phosphorylation events.

What emerging technologies are enhancing phospho-INSR detection sensitivity?

Recent technological advances are improving our ability to detect and quantify insulin receptor phosphorylation:

• Phosphoproteomics: Mass spectrometry-based approaches provide comprehensive phosphorylation profiling with site-specific resolution, allowing simultaneous detection of multiple phosphorylation events on INSR.

• Single-cell phospho-analysis: Emerging techniques enable phosphorylation analysis at the single-cell level, revealing cell-to-cell heterogeneity in insulin signaling.

• Phospho-specific biosensors: Genetically encoded FRET-based biosensors allow real-time monitoring of INSR phosphorylation dynamics in living cells.

• Proximity ligation assays: These techniques provide enhanced sensitivity for detecting phosphorylated proteins in tissue sections with spatial resolution.

How might Phospho-INSR (Tyr1361) Antibody contribute to precision medicine approaches?

Phospho-INSR (Tyr1361) Antibody has potential applications in developing precision medicine approaches for metabolic disorders:

• Patient stratification: Identifying subgroups of patients with specific defects in insulin receptor phosphorylation may guide personalized treatment strategies.

• Therapeutic monitoring: Using phospho-specific antibodies to monitor treatment response at the molecular level may help optimize therapeutic interventions.

• Drug development: Screening compounds for their ability to restore normal Tyr1361 phosphorylation patterns in patient-derived samples could accelerate the development of targeted therapies.

• Biomarker development: Incorporating phosphorylation status into multi-parameter biomarker panels may improve diagnostic and prognostic capabilities.