INSR (Ab-1375) Antibody

What is INSR (Ab-1375) Antibody and what epitope does it recognize?

INSR (Ab-1375) Antibody is a rabbit-derived polyclonal antibody that recognizes and binds to the insulin receptor (INSR). This antibody specifically targets a synthesized non-phosphopeptide derived from human Stathmin around the phosphorylation site of threonine 1375 (I-L-T(p)-L-P) . The antibody detects endogenous levels of total INSR protein, which is also known by alternative names including CD220 antigen, insulin receptor (IR), and kinase InsR .

The antibody has been affinity-purified from rabbit antiserum through affinity-chromatography using an epitope-specific immunogen, ensuring high specificity for the target protein . Understanding this epitope specificity is critical for experimental design, as it determines which regions of the INSR will be detectable using this antibody.

What are the recommended storage and handling conditions for maintaining antibody activity?

For optimal performance of INSR (Ab-1375) Antibody, researchers should adhere to the following storage and handling guidelines:

• Store the antibody at -20°C to maintain its stability and activity over time

• The antibody is supplied at a concentration of 1.0mg/ml in a formulation consisting of rabbit IgG in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol

• Avoid repeated freeze-thaw cycles as this can diminish antibody performance, similar to other antibody products that show reduced stability under such conditions

• Prior to use, allow the antibody to equilibrate to room temperature, and gently mix (do not vortex) to ensure homogeneity

• When diluting for experiments, use appropriate buffer systems that maintain the antibody's native conformation

Proper handling is critical as antibody degradation can lead to inconsistent results and reduced sensitivity in experimental applications.

What is the molecular weight of the INSR protein detected by this antibody?

The INSR protein detected by the INSR (Ab-1375) Antibody has an SDS-PAGE molecular weight of approximately 130kDa . This corresponds to the mature β subunit of the insulin receptor as observed in Western blot applications. Researchers should expect to see a band at this molecular weight when using the antibody for immunoblotting.

The insulin receptor is encoded by the INSR gene (Gene ID: 3643; UniProt accession: P06213) , and understanding its molecular weight is essential for proper identification in experimental results. The receptor is a heterotetramer consisting of two α and two β subunits, with the β subunit containing the transmembrane domain and tyrosine kinase activity.

What are the validated applications for INSR (Ab-1375) Antibody in research?

The primary validated application for INSR (Ab-1375) Antibody is Western blot (WB) . When using this antibody for Western blotting, researchers should follow these methodological guidelines:

• Sample preparation: Properly lyse cells or tissues in an appropriate buffer containing protease inhibitors

• Protein separation: Use SDS-PAGE with appropriate percentage gels (typically 7-10% for proteins of this size)

• Transfer: Transfer proteins to a membrane (PVDF or nitrocellulose) using standard protocols

• Blocking: Block non-specific binding sites with appropriate blocking buffer (typically 5% non-fat dry milk or BSA)

• Primary antibody incubation: Dilute INSR (Ab-1375) Antibody according to manufacturer's recommendations (typically 1:1000 to 1:2000)

• Detection: Use appropriate secondary antibodies and detection systems

While WB is the validated application, researchers interested in using this antibody for other applications such as immunoprecipitation, immunohistochemistry, or flow cytometry should perform validation studies to determine its suitability for these methods.

How should researchers optimize Western blot protocols when using INSR (Ab-1375) Antibody?

For optimal Western blot results with INSR (Ab-1375) Antibody, researchers should consider the following optimization strategies:

• Titration of antibody concentration: Test various dilutions to determine the optimal signal-to-noise ratio

• Incubation time and temperature: Typically, overnight incubation at 4°C provides better results than shorter incubations at room temperature

• Blocking reagent selection: Compare different blocking agents (BSA vs. non-fat dry milk) as they may affect background and specific signal differently

• Sample preparation optimization: Ensure complete denaturation and reduction of samples for optimal epitope exposure

• Signal detection system selection: Choose chemiluminescence, fluorescence, or colorimetric detection based on needed sensitivity

When troubleshooting poor results, consider the following common issues:

• High background: Increase blocking time, use more stringent washing, or further dilute the antibody

• Weak signal: Increase antibody concentration, protein loading, or exposure time

• Non-specific bands: Optimize blocking, increase antibody specificity with longer washes, or consider using gradient gels

What controls should be included when using INSR (Ab-1375) Antibody?

Proper experimental controls are essential when working with antibodies to ensure result validity. For INSR (Ab-1375) Antibody, researchers should include:

• Positive control: Lysates from cells known to express INSR (e.g., HepG2, 3T3-L1 adipocytes)

• Negative control: Lysates from cells with low or no INSR expression, or INSR-knockout cells

• Loading control: Detection of housekeeping proteins (e.g., β-actin, GAPDH) to normalize for protein loading

• Secondary antibody control: Omitting primary antibody to detect non-specific binding of secondary antibody

• Peptide competition assay: Pre-incubation of antibody with the immunizing peptide to confirm specificity

These controls help validate that the observed signal is specific to INSR and not due to non-specific binding or technical artifacts.

How can INSR (Ab-1375) Antibody be used to study insulin receptor mutations and their impact on insulin signaling?

INSR (Ab-1375) Antibody can be a valuable tool for studying insulin receptor mutations and signaling pathways. Based on research with anti-INSR antibodies:

• Expression analysis: The antibody can be used to detect expression levels of wild-type versus mutant INSR in cell models

• Autophosphorylation studies: After immunoprecipitation, the antibody can help assess whether mutations affect receptor autophosphorylation

• Trafficking studies: Analysis of cell surface versus intracellular receptors can reveal trafficking defects in mutant receptors

• Comparative signaling: The antibody can help determine if mutations affect downstream signaling via pathways such as Akt or ERK1/2

Research has shown that certain INSR mutations (e.g., P193L, F248C, R252C, S323L, F382V, D707A, P1178L) demonstrate diminished maximal autophosphorylation response to insulin, ranging from 0 to 27% of wild-type response . Using this antibody, researchers can assess whether similar defects are present in their models.

What is known about the cross-reactivity of anti-INSR antibodies with mutant insulin receptors?

Cross-reactivity with mutant receptors is an important consideration when studying insulin receptor variants. Based on available research:

• Most anti-INSR antibodies maintain binding to mutant receptors with single amino acid substitutions

• Even mutations close to antibody epitopes may not abolish binding, as demonstrated with antibody 83-7, which maintained binding to multiple mutant receptors

• Binding may be preserved even when receptor function is compromised

In studies of various INSR mutations, all expressed mutant receptors bound to the tested antibodies, even though some mutations were located near antibody epitopes . Flow cytometry analysis showed right-shifted peaks for all mutants relative to control IgG, indicating preserved antibody binding despite receptor mutations .

This suggests that INSR (Ab-1375) Antibody may be useful for detecting various mutant forms of INSR, though researchers should validate this for specific mutations of interest.

How can researchers use anti-INSR antibodies as potential therapeutic tools for insulin receptoropathies?

Recent research has explored the potential of anti-INSR antibodies as therapeutic agents for insulin receptoropathies caused by loss-of-function mutations:

• Surrogate agonist activity: Some anti-INSR antibodies can act as insulin mimetics, activating mutant receptors with impaired insulin binding

• Differential pathway activation: Antibodies may preferentially activate certain downstream pathways (e.g., Akt over ERK1/2), which could be therapeutically advantageous

• Combination therapy: Antibodies combined with insulin may increase maximal receptor phosphorylation compared to insulin alone for certain mutations

The table below summarizes findings from studies on antibody activation of mutant insulin receptors:

While INSR (Ab-1375) Antibody has not been specifically tested for this application, these findings suggest potential research directions for investigating therapeutic applications of anti-INSR antibodies .

What are the potential cross-reactivity concerns with INSR (Ab-1375) Antibody?

When using INSR (Ab-1375) Antibody, researchers should be aware of potential cross-reactivity issues:

• Species cross-reactivity: The antibody is specifically validated for human INSR (Hu) , and cross-reactivity with other species should be empirically determined

• Isoform specificity: INSR exists in two isoforms (A and B) due to alternative splicing of exon 11, and researchers should determine if the antibody detects both isoforms equally

• Related receptors: The insulin receptor belongs to a family that includes the insulin-like growth factor 1 receptor (IGF1R), which shares structural homology and may show cross-reactivity

• Non-specific binding: In certain cell types or under specific conditions, non-specific binding may occur

To address these concerns, researchers should:

• Validate the antibody in their specific experimental system

• Include appropriate controls as discussed in section 2.3

• Consider peptide competition assays to confirm specificity

• Compare results with alternative anti-INSR antibodies

How should researchers interpret variability in INSR detection between different cell types or tissues?

Variability in INSR detection across different samples can stem from multiple factors:

• Expression level differences: INSR expression varies naturally between tissues (highest in liver, muscle, and adipose tissue)

• Isoform distribution: The ratio of INSR-A to INSR-B isoforms differs between tissues and may affect detection

• Post-translational modifications: Glycosylation, phosphorylation, or other modifications may mask epitopes

• Sample preparation effects: Different lysis methods may extract INSR with varying efficiency

• Protein-protein interactions: Association with other proteins may block antibody access to epitopes

When analyzing such variability, researchers should:

• Normalize detection based on total protein loading

• Compare results with antibodies targeting different INSR epitopes

• Consider complementary methods like qRT-PCR to assess INSR mRNA levels

• Document tissue/cell-specific extraction conditions that optimize detection

What stability and quality control tests should researchers perform before using INSR (Ab-1375) Antibody in critical experiments?

Before using INSR (Ab-1375) Antibody in critical experiments, researchers should perform the following quality control tests:

• Functional validation: Test the antibody on positive control samples with known INSR expression

• Specificity testing: Perform peptide competition assays or test on INSR-knockout samples

• Lot-to-lot consistency: When receiving a new lot, compare performance with previously validated lots

• Sensitivity determination: Establish detection limits using dilution series of positive control samples

• Stability assessment: Evaluate antibody performance after various storage conditions and durations

For long-term projects, researchers should consider:

• Aliquoting antibody stock to minimize freeze-thaw cycles

• Periodically testing aliquots to ensure consistent performance

• Maintaining detailed records of antibody performance across experiments

• Using the same lot number for experiments requiring direct comparison

How can INSR (Ab-1375) Antibody be integrated into high-throughput screening approaches?

For high-throughput applications involving INSR (Ab-1375) Antibody, researchers can consider these methodological approaches:

• Automated Western blotting: Using capillary-based systems for higher throughput and reproducibility

• Reverse phase protein arrays (RPPA): Spotting multiple samples on arrays for parallel antibody probing

• Cell-based assays: Developing fixed-cell ELISA or high-content imaging workflows

• Multiplexed detection: Combining with other antibodies for simultaneous analysis of multiple proteins

When adapting for high-throughput use, consider these optimization steps:

• Determine minimum antibody concentration needed for reliable detection

• Establish robust positive and negative controls for each plate or batch

• Develop standardized protocols with minimal manual intervention

• Implement quality control metrics to flag problematic samples or wells

Similar approaches have been used successfully with other antibodies, as described in the literature where high-throughput antibody assessments were integrated with structural analyses to identify optimal antibody candidates .

How can INSR (Ab-1375) Antibody be used in conjunction with phospho-specific antibodies to study insulin signaling dynamics?

To study insulin signaling dynamics, INSR (Ab-1375) Antibody can be used alongside phospho-specific antibodies in the following approaches:

• Sequential immunoblotting: Probe first for phosphorylated INSR, then strip and reprobe with INSR (Ab-1375) to normalize phosphorylation to total receptor levels

• Dual-color Western blotting: Simultaneously detect total INSR and phospho-INSR using different fluorophore-conjugated secondary antibodies

• Immunoprecipitation-Western blot: Immunoprecipitate with INSR (Ab-1375) Antibody, then blot for phosphotyrosine or specific phospho-sites

• Proximity ligation assay: Combining INSR (Ab-1375) with phospho-specific antibodies to visualize phosphorylated receptor pools in situ

This approach allows researchers to:

• Quantify the proportion of receptors that become activated

• Track receptor activation kinetics after insulin stimulation

• Compare activation efficiency between wild-type and mutant receptors

• Assess the impact of drugs or genetic modifications on receptor phosphorylation

What are the considerations for using INSR (Ab-1375) Antibody in combination with other techniques to validate INSR-related findings?

• Genetic validation:

  • siRNA/shRNA knockdown of INSR

  • CRISPR-Cas9 editing to create INSR knockout or mutant lines

  • Rescue experiments with wild-type or mutant INSR expression constructs

• Functional validation:

  • Insulin binding assays

  • Glucose uptake measurements

  • Downstream signaling assessments (Akt, ERK phosphorylation)

  • Metabolic phenotyping (glycolysis, lipogenesis)

• Structural validation:

  • Mass spectrometry confirmation of INSR detection

  • Co-immunoprecipitation to verify protein interactions

  • Imaging techniques to confirm subcellular localization

• Cross-antibody validation:

  • Comparison with other anti-INSR antibodies targeting different epitopes

  • Epitope mapping to confirm binding specificity

This multi-faceted approach helps ensure that observations are not artifacts of a particular antibody or technique, providing more robust and reproducible findings.