INSRR Antibody

What is INSRR and what experimental applications benefit from INSRR antibodies?

INSRR (also known as IRR or Insulin Receptor-Related Receptor) is a transmembrane receptor belonging to the protein kinase superfamily, specifically within the tyrosine protein kinase family and insulin receptor subfamily . This receptor plays a role in insulin signaling and glucose metabolism, making INSRR antibodies valuable tools for:

• Characterizing insulin signaling pathways in various tissues

• Identifying Type B Intercalated Cells in kidney research

• Studying tyrosine kinase activity in receptor-based signaling

• Investigating phosphorylation of insulin receptor substrates IRS-1 and IRS-2

INSRR is a heterotetrameric receptor composed of two alpha and two beta chains linked by disulfide bonds . The protein has a calculated molecular weight of 144 kDa but is typically observed at 150-155 kDa in experimental contexts due to post-translational modifications .

Which tissues show significant INSRR expression for positive control samples?

When designing experiments requiring positive control samples for INSRR antibody validation, the following tissues have shown consistent INSRR expression:

For researchers seeking reliable positive controls, mouse or rat kidney tissue is most consistently cited across multiple antibody sources . Brain tissue (particularly human) can serve as an alternative positive control when kidney samples aren't available .

What are the optimal antibody dilutions for detecting INSRR across different experimental applications?

Optimal antibody dilutions vary depending on the specific antibody, application, and tissue type. Based on multiple antibody datasheets, the following ranges represent consensus recommendations:

It is strongly recommended that researchers titrate antibodies in their specific testing systems to determine optimal conditions for each experimental context . Western blot applications typically start with a 1:1000 dilution before optimization .

How should Western blot protocols be optimized for INSRR detection considering its observed molecular weight discrepancies?

INSRR protein has a calculated molecular weight of 144 kDa, but experimental observations show bands at 150-155 kDa or in some cases at 105 kDa . When optimizing Western blot protocols:

• Gel percentage selection: Use 6-8% gels for optimal separation in the high molecular weight range

• Extended transfer time: Implement 90-120 minute transfers at lower amperage for complete transfer of large proteins

• Blocking optimization: Use 5% BSA rather than milk for phosphorylated protein detection

• Sample preparation considerations:

  • Include phosphatase inhibitors if studying phosphorylated INSRR

  • Consider deglycosylation treatments to assess contribution of glycosylation to observed weight

• Positive controls: Run known positive samples (mouse kidney) alongside experimental samples

• Molecular weight markers: Use high-range markers that extend beyond 200 kDa

The discrepancy between calculated and observed molecular weights likely stems from post-translational modifications, particularly glycosylation and phosphorylation of the receptor .

How can researchers distinguish between specific and non-specific bands when detecting INSRR via Western blot?

When troubleshooting Western blot results for INSRR detection:

• Expected band size: Primary band should appear at 150-155 kDa for full-length INSRR, though some antibodies may detect a 105 kDa band depending on the epitope recognized

• Multiple band pattern analysis:

  • Alpha subunit: Extracellular portion, can appear as a separate band

  • Beta subunit: Contains transmembrane and cytoplasmic kinase domains

  • Proteolytic processing: INSRR undergoes processing to generate mature disulfide-linked α₂/β₂ tetrameric receptor

• Validation approaches:

  • Use tissue-specific positive controls (kidney, brain) alongside experimental samples

  • Run pre-adsorption controls with immunizing peptide when available

  • Compare results with multiple INSRR antibodies targeting different epitopes

  • Consider knockdown/knockout validation if possible

• Sample preparation factors:

  • Different protein extraction methods can yield different banding patterns

  • Post-translational modifications can significantly alter migration patterns

  • Different modified forms may appear as multiple bands simultaneously

Careful interpretation of Western blot results should consider the specific epitope recognized by the antibody, which may target portions of the alpha or beta subunit, or regions spanning both.

What are the most common causes of false negative results when using INSRR antibodies?

When investigating negative results with INSRR antibodies, consider:

• Sample-related issues:

  • Insufficient protein expression in selected samples

  • Protein degradation during sample preparation

  • Ineffective protein extraction from membrane fractions (INSRR is a membrane protein)

• Technical factors:

  • Suboptimal antibody dilution (start with 1:500 for WB when troubleshooting)

  • Inadequate antigen retrieval for IHC/ICC applications

  • Insufficient transfer of high molecular weight proteins

  • Incorrect secondary antibody selection

• Antibody-specific considerations:

  • Epitope masking due to protein folding or interactions

  • Epitope destruction during sample processing

  • Limited cross-reactivity with species being studied

• Storage and handling:

  • Antibody degradation due to improper storage

  • Repeated freeze-thaw cycles affecting antibody performance

  • Expired reagents or buffers

When troubleshooting, systematically evaluate each potential factor beginning with the most fundamental aspects of the protocol before proceeding to more complex considerations.

How can researchers effectively study the phosphorylation state of INSRR and its relation to downstream signaling?

Studying INSRR phosphorylation requires specialized approaches:

• Antibody selection: Use phospho-specific antibodies targeting key tyrosine residues when available, or pan-phosphotyrosine antibodies combined with INSRR immunoprecipitation

• Experimental design for phosphorylation analysis:

  • Maintain samples at 4°C with phosphatase inhibitors during preparation

  • Include positive controls (insulin-stimulated samples for related phosphorylation patterns)

  • Consider time-course experiments to capture transient phosphorylation events

• Downstream signaling analysis:

  • INSRR phosphorylates insulin receptor substrates IRS-1 and IRS-2

  • Monitor AKT1/PKB pathway activation, which is activated by INSRR signaling

  • Consider analysis of related pathways including cAMP-dependent PKA and actin nucleation by ARP-WASP complex

• Advanced techniques:

  • Proximity ligation assays to detect INSRR interactions with downstream effectors

  • Phosphoproteomics approaches for unbiased phosphorylation site mapping

  • FRET-based biosensors for real-time phosphorylation monitoring in living cells

Understanding the phosphorylation dynamics of INSRR provides crucial insights into its activation mechanisms and downstream signaling consequences in various physiological contexts.

What experimental approaches can help identify potential physiological ligands for INSRR?

INSRR is described as not binding known ligands of IR and IGF-1R despite high homology . To identify potential physiological ligands:

• Receptor binding assays:

  • Use purified INSRR extracellular domain for direct binding screens

  • Create INSRR-Fc fusion proteins for pull-down experiments

  • Develop competitive binding assays against known insulin/IGF ligands

• Cell-based approaches:

  • Reporter cell lines expressing INSRR and downstream signaling readouts

  • Comparative signaling studies with IR and IGF-1R

  • Tissue-specific conditioned media screening, particularly from tissues with high INSRR expression

• In silico analysis:

  • Structural modeling of INSRR binding pocket compared to IR/IGF-1R

  • Molecular docking studies with potential ligand candidates

  • Evolutionary analysis to identify conserved binding regions

• Physiological context investigation:

  • Focus on pH-dependent activation mechanisms, as INSRR has been suggested to function as an alkali sensor

  • Examine INSRR activity in tissues that experience pH fluctuations, particularly kidney

These approaches, used in combination, may help identify the elusive physiological ligands or activation mechanisms for INSRR, advancing our understanding of its biological functions.

How can INSRR antibodies be effectively employed to study Type B Intercalated Cells in kidney research?

INSRR has been identified as a marker for Type B Intercalated Cells in kidney research . For researchers in this field:

• Experimental approaches for identification and isolation:

  • Immunohistochemistry protocols optimized for kidney tissue sections

  • Multi-color immunofluorescence combining INSRR with other known markers

  • FACS-based cell sorting using membrane-targeted INSRR antibodies

• Co-localization studies:

  • Combine INSRR antibodies with other Type B Intercalated Cell markers

  • Use confocal microscopy for high-resolution co-localization analysis

  • Quantitative assessment of marker overlap in different physiological conditions

• Functional studies:

  • Correlate INSRR expression with cellular functions like pH regulation

  • Investigate changes in INSRR-positive cell populations in disease models

  • Consider ex vivo kidney slice cultures to maintain native cell architecture

• Technical considerations:

  • Optimize antigen retrieval methods for kidney tissue

  • Consider IHC-p (immunohistochemistry on paraffin sections) validated antibodies

  • Use fresh-frozen sections when possible for optimal epitope preservation

These methodological approaches enable researchers to leverage INSRR antibodies effectively for studying the specialized Type B Intercalated Cells in the kidney, advancing our understanding of renal physiology and pathophysiology.

What are the essential validation steps for confirming INSRR antibody specificity?

Before conducting extensive experiments, researchers should validate INSRR antibody specificity through:

• Positive and negative control tissues:

  • Positive controls: Mouse/rat kidney, human brain tissue

  • Negative controls: Tissues with negligible INSRR expression or INSRR-knockout samples

• Multi-technique validation:

  • Compare Western blot results with immunohistochemistry findings

  • Verify subcellular localization (membrane localization expected)

  • Cross-validate with mRNA expression data when possible

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide when available

  • Observe elimination of specific signal in positive control samples

• Multiple antibody verification:

  • Compare results with different antibodies targeting different INSRR epitopes

  • Use monoclonal and polyclonal antibodies for comparative analysis

• Knockdown validation when possible:

  • siRNA/shRNA-mediated knockdown should reduce antibody signal

  • CRISPR/Cas9 knockout provides the most definitive validation

Proper validation ensures experimental results accurately reflect INSRR biology rather than non-specific antibody interactions, which is particularly important given INSRR's homology to other insulin receptor family members.

How should researchers choose between monoclonal and polyclonal INSRR antibodies for different applications?

The choice between monoclonal and polyclonal INSRR antibodies depends on specific research goals:

For critical experiments:

• Western blot for protein quantification: Consider monoclonal antibodies for consistent results

• Immunoprecipitation: Polyclonal antibodies often perform better

• Cross-species studies: Polyclonal antibodies typically offer better cross-reactivity

• Detection of modified INSRR: Polyclonal antibodies may detect multiple forms

• Reproducible assay development: Monoclonal antibodies provide more consistent results

Many researchers use both types complementarily, validating findings with both monoclonal and polyclonal antibodies targeting different epitopes for maximum confidence in results.

How can INSRR antibodies contribute to understanding insulin resistance and metabolic disorders?

While INSRR doesn't bind classic insulin or IGF-1 ligands , its role in metabolic regulation offers research opportunities:

• Comparative expression analysis:

  • Quantify INSRR expression changes in diabetic vs. healthy tissues

  • Examine correlation between INSRR levels and insulin resistance markers

  • Investigate tissue-specific regulation in metabolic disease models

• Signaling pathway integration:

  • Study INSRR phosphorylation of IRS-1 and IRS-2 in health and disease

  • Investigate AKT1/PKB pathway activation by INSRR in metabolic contexts

  • Compare signaling outputs between INSRR and classic insulin receptor

• Methodological approaches:

  • Tissue microarrays with INSRR antibodies for high-throughput analysis

  • Phospho-specific detection of activated INSRR signaling

  • Co-immunoprecipitation studies to identify differential binding partners

• Therapeutic implications:

  • Screen for compounds that modulate INSRR activity

  • Evaluate INSRR as a potential compensatory mechanism in insulin resistance

  • Investigate INSRR-targeted approaches for metabolic intervention

These research directions may reveal previously unexplored roles for INSRR in metabolic regulation and potential therapeutic avenues for metabolic disorders.

What are the current methodological challenges in studying INSRR in neurological contexts?

INSRR is expressed in brain tissue , but studying its neurological functions presents unique challenges:

• Technical barriers:

  • Blood-brain barrier limitations for in vivo antibody applications

  • Complexity of neural cell types requiring precise localization

  • Protein extraction challenges from lipid-rich brain tissue

• Experimental approaches:

  • Brain slice immunohistochemistry with optimized antigen retrieval

  • Primary neuron cultures for functional studies

  • Cell-type specific analysis using co-localization with neuronal markers

• Methodological solutions:

  • Use fresh-frozen brain sections rather than fixed tissue when possible

  • Optimize protein extraction with specialized brain tissue lysis buffers

  • Consider stereotactic antibody delivery for in vivo studies

• Emerging techniques:

  • Single-cell analysis of INSRR expression in neural populations

  • Spatial transcriptomics combined with INSRR protein detection

  • Optogenetic approaches combined with INSRR pathway monitoring

Understanding INSRR's role in neurological contexts may provide insights into insulin signaling in the brain and potential connections to neurodegenerative conditions associated with insulin resistance.