IGFBP2 Antibody

What is IGFBP2 and what are its primary biological functions?

IGFBP2 is a 34.8 kDa protein belonging to the insulin-like growth factor binding protein family. It functions primarily as a regulator of IGF bioavailability and activity in the extracellular environment. IGFBP2 binds to IGF1 and IGF2 (with higher affinity for IGF2) and serves as a chaperone to facilitate their transport to target tissues . This protein plays crucial roles in multiple cellular processes including proliferation, differentiation, and apoptosis in a cell-type specific manner .

Beyond its canonical IGF-dependent functions, IGFBP2 exhibits several IGF-independent activities. It promotes cell migration through interaction with integrin alpha5 (ITGA5) via an RGD motif and enhances adhesion of endothelial progenitor cells to endothelial cells through ITGA5/ITGB1 interaction . IGFBP2 also functions coordinately with receptor protein tyrosine phosphatase beta/PTPRB and the IGF1 receptor to regulate IGF1-mediated signaling by promoting PTEN phosphorylation, which leads to AKT1 activation .

What types of IGFBP2 antibodies are available for research applications?

Research laboratories can utilize several types of IGFBP2 antibodies, each with specific applications:

• Monoclonal antibodies: Highly specific antibodies targeting particular epitopes, such as the C-10 clone that detects IGFBP2 protein of human origin through various applications including western blotting, immunoprecipitation, immunofluorescence, and ELISA . The EPR18012-257 clone is another well-characterized monoclonal option .

• Polyclonal antibodies: These recognize multiple epitopes on IGFBP2 protein, providing potentially higher sensitivity but possibly lower specificity than monoclonals .

• Species-specific antibodies: Available with reactivity to human, mouse, rat, and other species' IGFBP2, allowing for comparative studies across model organisms .

• Conjugated antibodies: Available in various forms including unconjugated, agarose-conjugated (for immunoprecipitation), HRP-conjugated (for direct detection in Western blots), and fluorophore-conjugated versions (FITC, PE, Alexa Fluor®) for flow cytometry and immunofluorescence applications .

• Neutralizing antibodies: Specifically designed to block IGFBP2 function in experimental settings, enabling functional studies of IGFBP2's biological roles .

How should I validate the specificity of IGFBP2 antibodies?

Thorough validation is critical to ensure reliable research results with IGFBP2 antibodies:

• Positive and negative controls:

  • Include cell lines or tissues known to express IGFBP2 as positive controls

  • Use IGFBP2 knockdown samples created with shRNA or siRNA approaches as negative controls

  • Include isotype-matched control antibodies (e.g., Rabbit monoclonal IgG [EPR25A]) to assess non-specific binding

• Multiple detection methods:

  • Confirm findings using different techniques (Western blot, IHC, ICC/IF)

  • Validate cellular localization patterns using subcellular fractionation followed by Western blotting

• Genetic approaches:

  • Use IGFBP2 knockdown/knockout samples to confirm antibody specificity

  • Compare protein detection with mRNA expression levels

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with other IGFBP family members

  • Consider using multiple antibodies targeting different epitopes

• Immunoprecipitation followed by mass spectrometry:

  • For definitive identification of the immunoprecipitated protein as IGFBP2

What are the optimal conditions for using IGFBP2 antibodies in Western blotting?

For successful Western blot detection of IGFBP2, consider the following optimized protocol:

Sample preparation:

• Use appropriate lysis buffers that preserve protein integrity

• Include protease inhibitors to prevent degradation

• Denature samples properly, considering IGFBP2's predicted molecular weight of 35 kDa

Blocking and antibody incubation:

• 5% non-fat dry milk in TBST is an effective blocking agent

• Antibody dilutions vary by product; for optimal results, follow manufacturer recommendations

• Incubate with primary antibody overnight at 4°C

Controls and validation:

• Include positive control samples known to express IGFBP2

• Use isotype control antibodies to assess non-specific binding

• Include IGFBP2 knockdown samples when possible

Detection and troubleshooting:

• For enhanced sensitivity, use HRP-conjugated secondary antibodies with chemiluminescent detection

• If detecting multiple bands, consider these may represent IGFBP2 fragments resulting from proteolysis

What are the recommended protocols for immunohistochemical detection of IGFBP2?

For optimal immunohistochemical detection of IGFBP2 in tissue sections:

Tissue preparation:

• Fix tissues appropriately (4% paraformaldehyde is commonly used)

• For paraffin-embedded sections, perform proper deparaffinization and rehydration

Antigen retrieval:

• Heat-induced epitope retrieval using Tris-EDTA buffer (pH 9.0) is recommended for optimal antigen accessibility

• Optimize retrieval time and temperature for specific tissues

Blocking and antibody incubation:

• Block with 5% goat serum to minimize non-specific binding

• Antibody dilutions vary by product; for example, ab188200 has been successfully used at 1:200 dilution

• Incubate primary antibody overnight at 4°C

Detection systems:

• For chromogenic detection, use appropriate HRP-conjugated secondary antibody followed by DAB color development

• For fluorescent detection, use fluorophore-conjugated secondary antibodies such as Alexa Fluor® 488

Controls:

• Include positive control tissues (e.g., choroid plexus for mouse brain samples)

• Use isotype control antibodies on serial sections

• Include a secondary-only control to assess background

How can IGFBP2 antibodies be effectively used in flow cytometry?

For successful flow cytometric analysis of IGFBP2 expression:

Cell preparation:

• Fix cells with 4% paraformaldehyde

• Permeabilize with an appropriate agent (90% methanol has been successfully used)

Antibody staining:

• Use antibodies validated for flow cytometry

• Optimal dilutions vary; for example, EPR18012-257 clone has been used at 1/60 dilution

• Include appropriate isotype controls matched to the primary antibody

Controls and validation:

• Include a negative control (cells without primary antibody incubation)

• Use cells with known IGFBP2 expression levels as positive controls

• Consider IGFBP2 knockdown cells as biological negative controls

Gating strategy:

• Exclude debris and doublets

• Use viability dyes to exclude dead cells

• When examining immune cell populations, include additional markers to identify specific cell types

Data analysis:

• Report results as mean/median fluorescence intensity

• Compare to appropriate isotype controls

• Consider dual staining with other markers to identify specific cell populations expressing IGFBP2

How can IGFBP2 antibodies be used to investigate its role in cancer progression?

IGFBP2 antibodies have become essential tools for exploring this protein's complex roles in cancer:

Expression profiling:

• Use IHC with IGFBP2 antibodies to characterize expression patterns across cancer types and correlate with clinical outcomes

• IGFBP2 overexpression has been associated with glioblastoma progression and poor patient survival

Functional studies:

• Apply neutralizing IGFBP2 antibodies (2.5-8 µg/ml) to cancer cell lines to study effects on proliferation, migration, and invasion

• Compare results with genetic knockdown approaches (shRNA or siRNA)

Signaling pathway analysis:

• Combine IGFBP2 antibodies with phospho-specific antibodies to study downstream signaling

• IGFBP2 has been shown to activate STAT3 signaling in cancer cells

Tumor microenvironment studies:

• Use multi-color immunofluorescence with IGFBP2 and immune cell markers to study spatial relationships

• IGFBP2 inhibition relieves immunosuppression by increasing CD8+ T and CD19+ B cells while decreasing CD163+ M2 macrophages in glioblastoma models

Biomarker development:

• Quantify IGFBP2 in patient samples using ELISA or mass spectrometry-based approaches

• Combined with other markers, IGFBP2 improves diagnostic performance for early-stage pancreatic cancer compared to CA19-9 alone

How can neutralizing IGFBP2 antibodies be utilized in functional studies?

Neutralizing antibodies provide powerful tools for interrogating IGFBP2 function:

Experimental design:

• Treat cells with neutralizing antibodies in serum-free medium to avoid interference

• Use concentration ranges of 2.5-8 µg/ml, as has been reported in published studies

• Include appropriate control IgG at equivalent concentrations

Functional readouts:

• Cell proliferation assays (MTT, BrdU incorporation)

• Colony formation assays

• Migration and invasion assays

• Signaling pathway activation (Western blotting for phosphorylated proteins)

Validation approaches:

• Compare effects with IGFBP2 genetic knockdown

• Assess specificity by attempting to rescue phenotypes with recombinant IGFBP2

• Confirm neutralization by measuring downstream signaling events

Combination strategies:

• Combine IGFBP2 neutralization with inhibitors of downstream pathways

• For example, targeting both IGFBP2 and STAT3 could provide insights into their relationship in promoting cell migration and proliferation

In vivo applications:

• Administration of neutralizing antibodies in animal models

• Blocking IGFBP2 has been shown to suppress tumor growth and improve survival in mouse glioblastoma models

How should I design experiments to distinguish between IGF-dependent and IGF-independent functions of IGFBP2?

Differentiating between IGFBP2's IGF-dependent and IGF-independent functions requires careful experimental design:

Antibody-based approaches:

• Use neutralizing antibodies targeting different IGFBP2 domains:

  • Antibodies targeting the IGF-binding domain

  • Antibodies targeting the RGD motif that mediates integrin binding

  • Compare effects to determine domain-specific functions

Genetic approaches:

• Generate IGFBP2 mutants with impaired IGF binding but intact integrin binding capacity

• Create IGFBP2 mutants with disrupted RGD motif but normal IGF binding

• Compare functional effects of these mutants to wild-type IGFBP2

Pathway analysis:

• Monitor both IGF receptor signaling (IGF1R/IR phosphorylation, IRS1/2 activation)

• Simultaneously assess alternative pathways (integrin signaling, FAK/Src activation)

• IGFBP2 has been shown to promote invasion and proliferation through integrin interactions independent of IGF binding

Co-factor considerations:

• IGFBP2 functions cooperatively with receptor protein tyrosine phosphatase beta (PTPRB) and IGF1R

• Assess the presence and role of such co-factors in your experimental system

Context dependency:

• The relative importance of IGF-dependent versus independent functions may vary by:

  • Cell type

  • Growth conditions

  • Pathological state (normal vs. cancer cells)

What are common issues encountered with IGFBP2 antibodies and how can they be resolved?

Researchers working with IGFBP2 antibodies frequently encounter these challenges:

Non-specific binding:

• Problem: Multiple unexpected bands in Western blot or non-specific tissue staining

• Solution: Optimize blocking (5% NFDM/TBST recommended) , increase antibody specificity by using monoclonals, include appropriate controls including isotype controls

Weak or absent signal:

• Problem: Insufficient detection despite expected IGFBP2 presence

• Solution: Optimize antibody concentration, improve antigen retrieval for IHC (Tris-EDTA buffer, pH 9.0) , consider more sensitive detection systems, ensure sample preparation preserves IGFBP2 integrity

Inconsistent results between experiments:

• Problem: Variable antibody performance across experiments

• Solution: Standardize protocols, maintain consistent incubation times and temperatures, prepare fresh working solutions, validate each new antibody lot

Discrepancies between antibodies:

• Problem: Different results obtained with different IGFBP2 antibodies

• Solution: Map epitopes recognized by each antibody, consider whether biological conditions might affect epitope accessibility, validate findings using multiple detection methods

IGFBP2 proteolytic processing:

• Problem: Detection of multiple fragments rather than full-length protein

• Solution: Use protease inhibitors during sample preparation, analyze with antibodies targeting different IGFBP2 regions, interpret multiple bands in context of known IGFBP2 processing

How should I interpret IGFBP2 expression data in cancer studies?

When analyzing IGFBP2 expression data in cancer:

Contextual interpretation:

• IGFBP2 is often overexpressed in various cancers, but its role may be context-dependent

• In some contexts, IGFBP2 functions as a tumor suppressor through its ability to sequester IGFs

• In other contexts, particularly through IGF-independent mechanisms, IGFBP2 may promote tumor progression

Expression patterns:

• Consider both intensity and localization of IGFBP2 staining

• Nuclear versus cytoplasmic localization may have different biological implications

• Extracellular/secreted IGFBP2 may have distinct functions from intracellular protein

Correlation with clinical data:

• Analyze associations between IGFBP2 expression and:

  • Patient survival

  • Disease progression

  • Response to therapy

  • Cancer subtype

Epigenetic regulation:

• IGFBP2 expression can be regulated by promoter methylation

• Hypermethylation resulting in reduced IGFBP2 expression has been observed in 30% of squamous cell lung cancers, 40% of colorectal cancers, >70% of lung adenocarcinomas, and 75% of hepatomas

• Consider epigenetic mechanisms when interpreting expression data

Integration with other biomarkers:

• IGFBP2 may provide more valuable information when analyzed together with other markers

• For pancreatic cancer, combining CA19-9, IGFBP2, and IGFBP3 significantly improves diagnostic performance compared to CA19-9 alone

How can IGFBP2 antibodies be used to study its role in immunomodulation?

Recent evidence suggests IGFBP2 plays important roles in immune regulation:

Tumor immunology applications:

• Multiplex immunofluorescence with IGFBP2 antibodies alongside immune cell markers to characterize the tumor immune microenvironment

• IGFBP2 inhibition has been shown to relieve immunosuppression in glioblastoma by increasing CD8+ T and CD19+ B cells while decreasing CD163+ M2 macrophages

Mechanistic studies:

• Investigation of IGFBP2's association with FcγRIIB phosphorylation on immune cells

• Analysis of how IGFBP2-induced mesenchymal features in cancer cells contribute to immunosuppression

Experimental approaches:

• Flow cytometry with IGFBP2 antibodies to analyze expression in specific immune cell populations

• Neutralizing antibody studies to assess direct effects on immune cell function

• Co-culture experiments with cancer cells and immune cells to study IGFBP2-mediated interactions

Therapeutic implications:

• The finding that blocking IGFBP2 can relieve immunosuppression suggests potential for combining IGFBP2-targeting approaches with immunotherapies

• Antibody-based in vivo studies can help evaluate such combination approaches in preclinical models

What recent advances have emerged regarding IGFBP2's role in therapy resistance?

Emerging research highlights IGFBP2's involvement in therapy resistance mechanisms:

IGFBP2 in gefitinib resistance:

• Recent studies demonstrate that IGFBP2/ITGA5 promotes acquired gefitinib resistance through STAT3 activation

• This mechanism operates in resistance caused by non-EGFR secondary mutations

Experimental approaches:

• IHC analysis with anti-IGFBP2 (1:200, ab188200) antibodies to assess expression in resistant tumors

• Complementary staining for STAT3 and downstream targets such as CXCL1

Combination targeting strategies:

• Investigating whether IGFBP2 inhibition can resensitize resistant cancer cells to therapy

• Potential for combining IGFBP2 targeting with STAT3 pathway inhibitors

Biomarker development:

• Assessment of whether IGFBP2 levels could predict response to targeted therapies

• Evaluating IGFBP2 as a biomarker for resistance monitoring

How can IGFBP2 and anti-IGFBP2 autoantibodies be developed as clinical biomarkers?

IGFBP2 shows promise as a biomarker, particularly in combination with anti-IGFBP2 autoantibodies:

Quantification methodologies:

• ELISA-based detection of IGFBP2 protein in biological fluids

• Mass spectrometry-based selected reaction monitoring/multiple reaction monitoring (SRM/MRM) for precise IGFBP2 quantification in plasma

• Indirect ELISA assay for measuring autoantibodies against IGFBP2:

  • Coat plates with recombinant IGFBP2 protein (0.5 µg/mL)

  • Create standard curve using serially diluted purified human IgG

  • Incubate with plasma samples

  • Detect with appropriate secondary antibodies

Diagnostic and prognostic value:

• IGFBP2 and anti-IGFBP2 antibodies serve as "independent negative prognostic markers"

• In pancreatic cancer, IGFBP2 combined with CA19-9 and IGFBP3
  improved diagnostic performance with higher AUC values than CA19-9 alone

• IGFBP2 appears elevated in pancreatic cancer risk conditions like intraductal papillary mucinous neoplasms (IPMNs)

Analytical considerations:

• Normalize anti-IGFBP2 antibody levels to total IgG concentration in each plasma sample

• Perform assessments in triplicate across independent assays to ensure reliability

• Include appropriate controls and standards in each assay

Multi-marker approach:

• Studies show the combination of CA19-9, IGFBP2, and IGFBP3 is significantly more effective for pancreatic cancer diagnosis than CA19-9 alone

• This demonstrates the value of IGFBP2 as part of a biomarker panel rather than in isolation