Phospho-IGF1R (Tyr1161) Antibody

What is Phospho-IGF1R (Tyr1161) Antibody and what specific epitope does it recognize?

Phospho-IGF1R (Tyr1161) Antibody is a polyclonal antibody that specifically detects the insulin-like growth factor 1 receptor (IGF1R) only when phosphorylated at tyrosine 1161. The antibody recognizes the phosphopeptide sequence around the phosphorylation site, specifically D-I-Y(p)-E-T derived from human IGF-1R . This specificity allows researchers to monitor the activation state of IGF1R in various experimental contexts. The antibody is typically produced by immunizing rabbits with synthetic phosphopeptide and KLH conjugates, followed by affinity purification using epitope-specific phosphopeptide chromatography . Non-phospho specific antibodies are typically removed by additional chromatography using non-phosphopeptide .

What are the validated applications for Phospho-IGF1R (Tyr1161) Antibody?

Phospho-IGF1R (Tyr1161) Antibody has been validated for multiple research applications:

When selecting this antibody for your research, consider that positive controls for Western blot include 293 cells treated with insulin, while breast carcinoma tissue serves as a suitable positive control for IHC applications .

What is the biological significance of IGF1R phosphorylation at Tyr1161?

IGF1R is a receptor tyrosine kinase that mediates actions of insulin-like growth factor 1 (IGF1). It binds IGF1 with high affinity and IGF2 and insulin with lower affinity . Phosphorylation at Tyr1161 is a critical event in IGF1R activation.

When ligand binding occurs, it activates the receptor kinase, leading to receptor autophosphorylation at multiple tyrosine residues including Tyr1161 . This phosphorylation event is crucial as it:

• Contributes to receptor activation and downstream signaling cascade initiation

• Enables the recruitment of signaling adapter proteins including insulin-receptor substrates (IRS1/2), Shc, and 14-3-3 proteins

• Initiates two primary signaling pathways: the PI3K-AKT/PKB pathway and the Ras-MAPK pathway

• Plays a role in cell growth, survival control, and is crucial for tumor transformation and malignant cell survival

Monitoring phosphorylation at this specific residue provides insights into IGF1R activation status and potential downstream signaling activities in both normal physiology and pathological conditions.

What are the recommended protocols for using Phospho-IGF1R (Tyr1161) Antibody in Western blotting?

For optimal Western blot results with Phospho-IGF1R (Tyr1161) Antibody:

Sample Preparation:

• Use cell lysates from 293 cells treated with insulin as positive controls

• Brain and spleen lysates have also been validated as positive controls

• Prepare samples in standard RIPA or NP-40 lysis buffer with phosphatase inhibitors to preserve phosphorylation status

Protocol Recommendations:

• Load 20-50 μg protein per lane on SDS-PAGE (8-10% gel recommended)

• Transfer to PVDF or nitrocellulose membrane

• Block with 5% BSA in TBST (not milk, as it contains phosphatases)

• Dilute primary antibody (typically 1:1000) in blocking buffer

• Incubate overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit IgG)

• Develop using enhanced chemiluminescence (ECL)

The antibody should detect bands at approximately 95 and 200 kDa, corresponding to the beta subunit and full-length IGF1R, respectively .

How can I optimize immunohistochemistry and immunofluorescence protocols for the Phospho-IGF1R (Tyr1161) Antibody?

For successful IHC and IF applications with Phospho-IGF1R (Tyr1161) Antibody:

Immunohistochemistry (IHC-P):

• Deparaffinize sections in xylene and rehydrate through graded alcohols

• Perform heat-induced epitope retrieval with high-pH buffer for optimal antigen retrieval

• Block endogenous peroxidase with 3% H₂O₂

• Block with normal serum or protein blocking solution

• Incubate with 1:50 dilution of primary antibody

• Use a specific secondary antibody system (e.g., DAB Map detection kit)

• Counterstain with hematoxylin

• Score as positive when at least 1% of cells show cytoplasmic expression

Immunofluorescence (IF):

• Fix cells with 4% paraformaldehyde

• Permeabilize with 0.1-0.5% Triton X-100

• Block with normal serum in PBS with 1% BSA

• Incubate with primary antibody diluted 1:50-1:200

• Wash thoroughly with PBS

• Apply fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Co-staining experiments can be particularly informative; for example, co-staining with F-actin or vinculin can reveal IGF1R localization to focal adhesions and cellular protrusions .

What controls should be included when working with Phospho-IGF1R (Tyr1161) Antibody?

Including appropriate controls is critical for ensuring experimental validity:

Positive Controls:

• Cell lines: 293 cells treated with insulin , MCF-7 cells

• Tissues: Human breast carcinoma , brain and spleen lysates

• Recombinant proteins: Synthesized phosphopeptide corresponding to the target sequence

Negative Controls:

• Phosphatase-treated samples: Treat duplicate samples with lambda phosphatase to remove phosphorylation and demonstrate antibody specificity

• Blocking peptide: Pre-incubate antibody with phospho-peptide to block specific binding

• Non-phosphorylated control: Use samples where IGF1R is known to be unphosphorylated

• Isotype control: Use rabbit IgG at the same concentration as the primary antibody

Technical Controls:

• Include beta-actin or GAPDH as loading controls for Western blots

• For phospho-arrays, use the internal controls provided (beta-actin, GAPDH, negative controls)

How can the specificity of Phospho-IGF1R (Tyr1161) Antibody be validated in experimental systems?

Validating antibody specificity is essential for reliable research outcomes:

• Peptide competition assay: Pre-incubate the antibody with excess phospho-peptide before application to your samples. Signal loss confirms specificity for the phospho-epitope.

• Kinase inhibition: Treat cells with IGF1R kinase inhibitors (e.g., AG1024) to prevent phosphorylation, which should eliminate antibody binding .

• Phosphatase treatment: Treat lysates with lambda phosphatase to remove phosphate groups from all proteins. The antibody signal should disappear after this treatment.

• siRNA/CRISPR knockdown: Reduce IGF1R expression using genetic approaches. This should decrease or eliminate antibody signal, confirming target specificity.

• Cross-reactivity assessment: Test the antibody against related receptors like insulin receptor to ensure it doesn't detect similar phosphorylation sites. Some antibodies may detect both IR and IGF1R phosphorylation at equivalent sites (as seen with the dual-specific IR/IGF1R phospho-antibodies) .

• Stimulation experiments: Compare signal between unstimulated cells and cells treated with IGF-1 to demonstrate increased phosphorylation following receptor activation.

How is Phospho-IGF1R (Tyr1161) Antibody used to study epithelial-to-mesenchymal transition (EMT) in cancer research?

Phospho-IGF1R (Tyr1161) Antibody has provided valuable insights into the role of IGF1R activation in epithelial-to-mesenchymal transition (EMT), a critical process in cancer progression:

• Localization studies: Research has revealed that phospho-IGF1R (Tyr1161) shows distinct subcellular distribution during EMT. In cells undergoing EMT, active IGF1R localizes to cell membrane protrusions and focal adhesion-like structures . High-content immunofluorescence microscopy studies demonstrated striking cell membrane-associated local accumulation of phospho-IGF1R (Tyr1161) at the tips of numerous cell protrusions in cells with EMT-like phenotypes .

• Cytoskeletal reorganization: Co-staining experiments with phospho-IGF1R (Tyr1161) and cytoskeletal proteins (F-actin, vinculin) have shown that IGF1R activation is associated with dramatic reorganization of the cytoskeleton during EMT, with F-actin stress fibers emanating from phospho-IGF1R-positive focal adhesion-like structures .

• Signaling pathway integration: Studies have demonstrated that TGFβ1-induced EMT proceeds through IGF1R activation. Treatment with TGFβ1 causes remarkable reorganization of active IGF1R at the tips of prominent F-actin stress fibers, which can be prevented by IGF1R inhibitors like AG1024 .

• Therapeutic resistance mechanisms: Research using phospho-IGF1R (Tyr1161) antibody has shown that erlotinib-resistant cancer cells display EMT-like features with enhanced IGF1R signaling and redistribution of phosphorylated IGF1R to focal adhesions .

These findings suggest that targeting IGF1R phosphorylation might be a strategy to prevent EMT and associated therapeutic resistance in cancer treatment.

What insights has Phospho-IGF1R (Tyr1161) Antibody provided in understanding resistance mechanisms to targeted therapies?

Phospho-IGF1R (Tyr1161) Antibody has been instrumental in elucidating mechanisms of resistance to targeted cancer therapies:

• EGFR-TKI resistance: Studies using phospho-IGF1R (Tyr1161) antibody demonstrated that erlotinib-refractory PC-9 cancer cells (with EGFR mutations) show enhanced IGF1R signaling with distinct subcellular localization of the phosphorylated receptor . This research revealed that IGF1R activation represents an alternative survival pathway when EGFR signaling is blocked.

• EMT-mediated resistance: Research showed that induction of EMT (via TGFβ1 treatment) proceeds through IGF1R activation, leading to erlotinib desensitization even in cells with EGFR mutations that would normally predict sensitivity . The half-maximal effective concentrations of erlotinib could be restored when TGFβ1-treated cells were co-treated with IGF1R inhibitor AG1024 .

• Cellular adaptation mechanisms: The subcellular distribution of phospho-IGF1R (Tyr1161) in resistant cells revealed its concentration at the edges of focal adhesion-like structures and cell protrusions, suggesting reorganization of signaling complexes as part of resistance mechanisms .

• Pathway cross-talk: The co-localization of phospho-IGF1R with cytoskeletal proteins during resistance development demonstrated how receptor tyrosine kinase signaling integrates with cellular structural changes to promote survival under therapeutic pressure .

These findings highlight the potential of dual targeting strategies (e.g., EGFR plus IGF1R inhibition) to overcome resistance to targeted therapies in certain cancers.

How can Phospho-IGF1R (Tyr1161) antibody be used in phospho-protein profiling and pathway analysis?

Phospho-IGF1R (Tyr1161) antibody is valuable for comprehensive phosphorylation profiling and integrated pathway analysis:

• Phospho-antibody arrays: Phospho-IGF1R (Tyr1161) antibody is incorporated into high-throughput antibody arrays such as the IGF1R Phospho Antibody Array for qualitative protein phosphorylation profiling . These arrays allow researchers to:

  • Compare normal samples to treated or diseased samples

  • Identify candidate biomarkers

  • Study multiple phosphorylation sites simultaneously

• Multi-parameter signaling analysis: When used in combination with antibodies against downstream effectors (e.g., phospho-AKT, phospho-ERK), the phospho-IGF1R antibody enables mapping of entire signaling cascades activated following receptor phosphorylation.

• Temporal signaling dynamics: By collecting samples at multiple time points after stimulation and analyzing phospho-IGF1R levels alongside other pathway components, researchers can elucidate the temporal dynamics of signal transduction.

• Cross-pathway integration: Including phospho-IGF1R (Tyr1161) in panels with other receptor tyrosine kinases (RTKs) and their downstream targets allows for analysis of pathway crosstalk and integration.

The IGF1R Phospho Antibody Array includes 245 site-specific and phospho-specific antibodies (with 6 replicates per antibody) to enable comprehensive profiling across multiple signaling nodes . This approach is particularly valuable for studying how IGF1R activation integrates with other pathways like AKT, MAPK, STAT, and mTOR signaling.

What are the challenges and solutions for using Phospho-IGF1R (Tyr1161) Antibody in tissue microarrays?

Tissue microarray (TMA) analysis with Phospho-IGF1R (Tyr1161) Antibody presents specific challenges and solutions:

Challenges:

• Tissue processing effects: Phospho-epitopes are particularly sensitive to fixation conditions and processing methods, which can affect antigen detection in TMAs.

• Phosphatase activity: Post-mortem and processing-related phosphatase activity can reduce phospho-IGF1R signal in tissue samples.

• Heterogeneity representation: The small tissue cores used in TMAs may not represent tumor heterogeneity adequately.

• Scoring standardization: Establishing consistent scoring methods for phospho-IGF1R positivity across different studies and operators.

Solutions and Methodological Approaches:

• Optimized TMA construction: Use multiple tissue cylinders (e.g., three cylinders with 1.0 mm diameter) from representative areas of each donor tissue block to create more representative TMAs .

• Epitope retrieval optimization: Employ heat-induced epitope retrieval with high-pH buffer specifically optimized for phospho-IGF1R (Tyr1161) detection .

• Standardized scoring system: Implement consistent scoring criteria, such as defining positivity when at least 1% of cells show cytoplasmic expression of phospho-IGF1R .

• Controls within TMAs: Include positive and negative control tissues within each TMA block to ensure consistent staining across batches.

• Digital image analysis: Utilize computerized image analysis to achieve more objective and reproducible quantification of phospho-IGF1R staining.

What are common technical issues when using Phospho-IGF1R (Tyr1161) Antibody and how can they be resolved?

When working with Phospho-IGF1R (Tyr1161) Antibody, researchers may encounter several technical challenges:

For Western blotting specifically:

• Prevent phospho-epitope loss by lysing cells directly in SDS sample buffer with phosphatase inhibitors

• Use freshly prepared samples whenever possible

• Consider using PVDF rather than nitrocellulose membranes for better retention of phospho-proteins

For immunohistochemistry/immunofluorescence:

• Process tissues quickly and use appropriate fixatives (10% neutral buffered formalin)

• Ensure tissue sections are not exposed to phosphatase-containing solutions

How can subcellular localization of phospho-IGF1R (Tyr1161) be accurately characterized?

Characterizing the subcellular localization of phospho-IGF1R (Tyr1161) requires specific approaches to maintain signal specificity while enabling detailed visualization:

• High-content immunofluorescence microscopy:

  • This approach has successfully visualized sub-cellular accumulation of phospho-IGF1R (Tyr1161) at cell membrane protrusions and focal adhesions

  • Use confocal microscopy with Z-stack acquisition for three-dimensional localization analysis

• Co-localization studies:

  • Perform co-staining with markers of specific cellular compartments:

    • F-actin for cytoskeletal association and cellular protrusions

    • Vinculin for focal adhesions

    • E-cadherin for cell-cell junctions

    • Membrane markers to distinguish surface vs. internalized receptor

• Sample preparation optimization:

  • Use mild permeabilization conditions to preserve membrane structure

  • Consider using super-resolution microscopy techniques for nanoscale localization

  • Apply phosphatase inhibitors throughout the fixation and staining process

• Dynamic localization studies:

  • Combine with live-cell imaging of fluorescently tagged IGF1R to track receptor movement before fixation and phospho-staining

  • Perform time-course experiments after stimulation to track phospho-IGF1R redistribution

• Biochemical fractionation validation:

  • Complement imaging with subcellular fractionation and Western blot analysis of phospho-IGF1R in different cellular compartments

  • Use this approach to validate microscopy findings with an independent method

Research has shown that phospho-IGF1R (Tyr1161) localization can be dynamically regulated during processes like EMT, with significant redistribution to cell protrusions and focal adhesions that correlates with cytoskeletal reorganization .

How can phospho-IGF1R (Tyr1161) detection be integrated with other analytical approaches for comprehensive research?

Integrating phospho-IGF1R (Tyr1161) detection with complementary analytical approaches enhances research depth:

• Multi-omics integration:

  • Combine phospho-IGF1R analysis with:

    • RNA-seq/qPCR to correlate receptor activation with transcriptional changes

    • Proteomics to identify novel interacting partners and pathway components

    • Metabolomics to link IGF1R signaling to metabolic adaptations

• Functional assays correlation:

  • Pair phospho-IGF1R detection with:

    • Cell proliferation and survival assays

    • Migration and invasion assessments for cancer research

    • Glucose uptake measurements to connect to metabolic functions

    • Drug sensitivity testing to correlate activation state with therapeutic response

• Advanced imaging combinations:

  • Integrate with:

    • Proximity ligation assay (PLA) to detect phospho-IGF1R interactions with specific partners

    • FRET/BRET approaches to study dynamics of IGF1R phosphorylation and protein interactions

    • Live-cell phospho-sensors to monitor IGF1R activation in real-time

• Cell-based ELISA applications:

  • Phospho-IGF1R (Tyr1161) Colorimetric Cell-Based ELISA kits allow for:

    • Quantitative assessment of phosphorylation levels in intact cells

    • High-throughput screening of compounds affecting IGF1R phosphorylation

    • Normalization to total IGF1R levels for accurate activation analysis

• Phospho-antibody arrays:

  • Use commercial arrays featuring phospho-IGF1R (Tyr1161) alongside multiple other phosphorylation sites

  • These arrays enable monitoring of entire signaling networks with up to 245 antibodies per array

  • Allow parallel analysis of phosphorylation events across multiple pathways

This integrated approach provides a systems-level understanding of IGF1R signaling and its biological consequences in both normal physiology and disease states.