INSRR Antibody, Biotin conjugated

What is INSRR and why are INSRR antibodies important in research?

INSRR, also known as IRR, belongs to the protein kinase superfamily, tyrosine protein kinase family, and insulin receptor subfamily. This heterotetrameric transmembrane receptor consists of two alpha and two beta chains linked by disulfide bonds. INSRR shares high homology with insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R), but interestingly doesn't bind to any of their known ligands .

INSRR demonstrates tyrosine-protein kinase activity and phosphorylates insulin receptor substrates IRS-1 and IRS-2. The receptor likely binds an insulin-related protein that hasn't been fully characterized . INSRR antibodies are crucial for studying this receptor's expression patterns, particularly in kidney tissues of mice and brain tissues of humans where positive Western blot detection has been confirmed .

What are the advantages of biotin-conjugated antibodies in immunoassay research?

Biotin-conjugated antibodies leverage the exceptional binding affinity between biotin and streptavidin/avidin (Kd ≈ 10^-15 M), which is among the strongest non-covalent interactions in biology . The key advantages include:

• Signal amplification capabilities, allowing researchers to use highly diluted primary antibodies

• Stability against proteolytic enzymes, pH fluctuations, temperature variations, and denaturing reagents

• Preservation of natural binding properties of antibodies and antigens through indirect interaction

• Versatility in application across multiple detection platforms

• Biotin's small size (240 Da) and flexible valeric side chain make it ideal for protein labeling without altering antibody-antigen interactions

How does biotin conjugation affect antibody function for INSRR detection?

When properly optimized, biotin conjugation preserves the functional activity of INSRR antibodies while providing enhanced detection capabilities. The conjugation occurs through the attachment of biotin molecules to amino groups of antibodies using biotinylation reagents. An optimal conjugation ensures:

• Retention of antigen recognition specificity to INSRR epitopes

• Minimal steric hindrance affecting antibody-antigen binding

• Appropriate biotin-to-antibody ratio that balances detection sensitivity with native antibody function

For INSRR antibodies specifically, careful optimization of the biotinylation process is essential since over-biotinylation can potentially interfere with binding to the target epitope regions such as the AA 668-702 sequence used for antibody generation .

What optimization steps are critical when preparing biotin-conjugated INSRR antibodies?

Optimization of biotin-conjugated INSRR antibodies requires careful consideration of several parameters:

Research shows that using 5 μg of antibody at 1 mg/ml concentration yields optimal results when testing reactivity with gold-conjugated streptavidin . For INSRR antibodies specifically, which target regions like AA 668-702, maintaining the structural integrity of these epitope-recognizing domains is crucial during conjugation.

How do anti-biotin antibodies compare to streptavidin for detection in INSRR studies?

Anti-biotin antibodies and streptavidin offer complementary approaches for INSRR detection systems:

Anti-biotin antibody approach:

• Enables unprecedented enrichment of biotinylated peptides from complex mixtures

• Provides direct, higher confidence detection of labeled proteins

• Identifies specific biotinylation sites with greater precision

• Yields significantly more biotinylation sites (30-fold increase) compared to streptavidin-based enrichment

Streptavidin-based approach:

• Provides broader identification of biotinylated proteins

• Can detect proteins where any non-biotinylated peptide meets statistical thresholds

• Often identifies more total proteins but with less site-specific information

For comprehensive INSRR studies, employing both methods provides complementary data. For example, when studying INSRR receptor phosphorylation patterns or protein interactions, anti-biotin antibodies would provide precise site identification, while streptavidin could capture a broader range of INSRR-associated proteins.

What interference issues should researchers address when using biotin-conjugated INSRR antibodies?

Several interference mechanisms can affect experiments using biotin-conjugated INSRR antibodies:

• Endogenous biotin interference: Biological samples may contain natural biotin that competes with biotinylated antibodies for streptavidin binding sites

• High-biotin sample interference: Elevated levels of supplemental biotin can cause either falsely elevated or suppressed test results in immunoassays

• Non-specific binding: Streptavidin may bind to biotin-like structures in complex samples

• Cross-reactivity with related receptors: Due to INSRR's high homology with insulin receptor (IR) and IGF-1R, careful validation is required to ensure specificity

To address these issues, researchers should:

• Include appropriate blocking steps using biotin-free blocking reagents

• Consider biotin-depleting pretreatment of samples when necessary

• Validate antibody specificity using knockout/knockdown controls

• Include competitive binding assays to confirm binding specificity to INSRR rather than related receptors

What is the recommended protocol for biotin-labeling INSRR antibodies?

Based on optimized protocols from related research, the following procedure is recommended for biotin-labeling INSRR antibodies:

• Preparation:

  • Dilute INSRR antibody to 1 mg/ml in PBS (pH 7.4)

  • Ensure antibody solution is free of amine-containing additives

• Conjugation:

  • Add 1 μl of modifier reagent to 10 μl of INSRR antibody solution (5 μg optimal concentration)

  • Mix gently and add to lyophilized Biotin Conjugation Mix

  • Incubate at room temperature in the dark for 20 minutes

• Quenching:

  • Add 1 μl of quencher reagent and mix gently

  • Allow reaction to stand for 10 minutes

• Purification (optional):

  • Dialyze against PBS or use size exclusion chromatography to remove excess biotin

• Storage:

  • Store at 4°C for short-term or aliquot and store at -20°C

  • Add 0.02% sodium azide and 50% glycerol for long-term storage

Research indicates that both freshly prepared and properly stored biotinylated antibodies show comparable reactivity when tested .

How should researchers validate biotin-conjugated INSRR antibodies before experimental use?

A comprehensive validation strategy includes:

• Western Blot Validation:

  • Confirm detection of INSRR at the expected molecular weight (144 kDa calculated, 150-155 kDa observed)

  • Test reactivity in known positive tissues (mouse kidney, human brain)

  • Compare detection with non-biotinylated antibody to assess impact of conjugation

• Specificity Testing:

  • Perform competitive binding assays

  • Test against knockout/knockdown samples

  • Cross-reactivity assessment with related receptors (IR, IGF-1R)

• Functional Assessment:

  • Verify retention of binding ability using ELISA

  • Assess signal-to-noise ratio in immunohistochemistry applications

• Biotin-to-Protein Ratio Determination:

  • HABA assay to quantify biotin incorporation

  • Ensure optimal labeling (3-8 biotin molecules per antibody typically)

What controls are essential when using biotin-conjugated INSRR antibodies in immunoassays?

How can researchers distinguish specific signals from background when using biotin-conjugated INSRR antibodies?

Distinguishing specific signals from background requires systematic analysis:

• Signal intensity quantification:

  • Normalize signal intensity against housekeeping proteins

  • Apply statistical thresholds for signal-to-noise ratio determination

  • Perform replicate analysis to establish consistency

• Background reduction strategies:

  • Optimize blocking conditions (5% BSA or milk protein in wash buffer)

  • Adjust streptavidin-HRP dilution (typically 1:1000-1:5000)

  • Include 0.1-0.5% Tween-20 in wash buffers to reduce non-specific binding

  • Consider using specially formulated low-background streptavidin reagents

• Comparative analysis:

  • Parallel testing with non-biotinylated INSRR antibody

  • Cross-validation with alternative detection methods

  • Side-by-side comparison of different tissues with varying INSRR expression levels

• Signal specificity confirmation:

  • Peptide competition assays using the immunizing peptide (AA 668-702)

  • Antibody dilution series to determine optimal working concentration (1:500-1:2000 for WB applications)

What are common pitfalls when working with biotin-conjugated INSRR antibodies and how can they be addressed?

How should researchers interpret and validate contradictory results from biotin-streptavidin versus conventional detection methods?

When facing contradictory results between biotin-streptavidin and conventional detection systems:

• Methodological validation:

  • Compare results from both streptavidin-based protein enrichment and anti-biotin antibody-based peptide enrichment, as these provide complementary information

  • Verify whether discrepancies arise from detection sensitivity differences rather than true biological variation

• Cross-platform confirmation:

  • Validate findings using orthogonal techniques (mass spectrometry, immunoprecipitation, functional assays)

  • Test if results are consistent across different sample types and experimental conditions

• Technical considerations:

  • Assess if biotin conjugation might be affecting the critical epitope regions of the INSRR antibody

  • Evaluate if the streptavidin system's high sensitivity is detecting physiologically irrelevant levels of INSRR

• Biological interpretation:

  • Consider if discrepancies reveal previously unknown INSRR isoforms or post-translational modifications

  • Examine if different detection methods are preferentially capturing different conformation states of the receptor

How can biotin-conjugated INSRR antibodies be utilized in proximity labeling experiments?

Proximity labeling with biotin-conjugated INSRR antibodies offers powerful insights into receptor interactions:

• APEX-based proximity labeling:

  • APEX peroxidase-mediated biotinylation followed by anti-biotin antibody enrichment can identify proteins in close proximity to INSRR

  • This approach yields significantly more biotinylation sites (>30-fold increase) compared to streptavidin-based protein enrichment

  • For INSRR studies, this can reveal transient interaction partners and signaling components

• BioID approach:

  • Fusion of BioID ligase to INSRR for proximity-dependent biotinylation

  • Allows identification of proteins within approximately 10 nm radius of INSRR

  • Particularly useful for mapping INSRR signaling complexes

• Spatial proteomics applications:

  • Subcellular localization studies of INSRR using compartment-specific markers

  • Investigation of INSRR trafficking between membrane and intracellular compartments

A typical protocol would include:

• Expression of APEX-tagged INSRR constructs

• Biotin-phenol labeling (typically 1 mM biotin-phenol, 1 mM H₂O₂, 1 minute)

• Cell lysis under denaturing conditions

• Enrichment of biotinylated peptides using anti-biotin antibodies rather than streptavidin

• Mass spectrometry analysis to identify interaction partners

What emerging technologies enhance the utility of biotin-conjugated INSRR antibodies?

Several cutting-edge technologies are expanding applications for biotin-conjugated INSRR antibodies:

• Modified lateral flow test systems:

  • Implementation of gold-streptavidin and biotinylated antibodies as detector probes

  • Shows improved sensitivity and specificity (>95% sensitivity, >99% specificity in comparable systems)

  • Potential for rapid INSRR detection in research and diagnostic applications

• Multiplexed detection platforms:

  • Simultaneous detection of INSRR alongside related receptors (IR, IGF-1R)

  • Utilizes differentially labeled streptavidin conjugates

  • Enables complex signaling pathway analysis in limited samples

• Super-resolution microscopy applications:

  • Biotin-streptavidin systems provide excellent signal amplification for nanoscale imaging

  • Allows visualization of INSRR clustering and membrane microdomain localization

  • Can be combined with proximity ligation assays to visualize protein interactions in situ

• Microfluidic and lab-on-chip platforms:

  • Integration of biotin-conjugated antibodies into automated microfluidic systems

  • Enables high-throughput analysis with minimal sample consumption

  • Particularly valuable for phosphorylation profiling of INSRR in response to stimuli