ITGB1BP1 Antibody

What is ITGB1BP1 and what functional significance does it have in cellular processes?

ITGB1BP1, also known as ICAP1, is a key regulator of integrin-mediated cell-matrix interaction signaling that functions by binding to the ITGB1 cytoplasmic tail. It prevents the activation of integrin alpha-5/beta-1 (heterodimer of ITGA5 and ITGB1) by competing with activators like talin or FERMT1 .

The protein plays significant roles in:

• Cell proliferation, differentiation, spreading, adhesion, and migration

• Mineralization and bone development

• Angiogenesis regulation

• Focal adhesion dynamics

• Rho GTPase signaling

ITGB1BP1 has been identified as a negative regulator of angiogenesis by attenuating endothelial cell proliferation and migration, lumen formation, and sprouting angiogenesis. This occurs through promoting AKT phosphorylation and inhibiting ERK1/2 phosphorylation via activation of the Notch signaling pathway .

What applications are most reliable for ITGB1BP1 antibody detection and what are their optimal working dilutions?

ITGB1BP1 antibodies have been validated for multiple applications with specific recommended dilutions:

For optimal results, it's recommended to perform a titration for each specific experimental system as effectiveness can be sample-dependent . Antigen retrieval methods may significantly impact staining quality in IHC applications, with many protocols suggesting TE buffer pH 9.0 or citrate buffer pH 6.0 .

How do polyclonal and monoclonal ITGB1BP1 antibodies differ in research applications?

Both polyclonal and monoclonal antibodies against ITGB1BP1 are available for research use, each with specific advantages:

Polyclonal Antibodies:

• Recognize multiple epitopes on ITGB1BP1, potentially increasing sensitivity

• Examples include rabbit polyclonal antibodies with reactivity to human, mouse, and rat samples

• Often purified through protein A columns followed by peptide affinity purification

• Suitable for multiple applications including WB, IHC, IF/ICC, and IP

Monoclonal Antibodies:

• Recognize a single epitope, potentially providing higher specificity

• Examples include mouse monoclonal antibodies like clone OTI6A12

• Offer consistent results between batches

• Particularly useful for applications requiring high specificity such as co-immunoprecipitation studies

When studying novel interactions or in complex tissue samples, polyclonal antibodies may offer advantages in detection sensitivity, while monoclonal antibodies provide greater consistency for quantitative studies and reproducibility between experiments .

What is the molecular weight of ITGB1BP1 and how can this be used to validate antibody specificity?

ITGB1BP1 has a calculated molecular weight of approximately 22 kDa (21.8 kDa precisely), which corresponds to its 200 amino acid sequence length . In Western blot analysis, ITGB1BP1 is typically observed as a band at 22 kDa .

To validate antibody specificity:

• Run positive control samples (e.g., PC-3 cells, mouse thymus tissue, or human brain tissue) alongside experimental samples

• Include negative controls such as knockout cell lines if available

• Compare the observed band with the predicted molecular weight (22 kDa)

• If multiple bands appear, perform additional validation such as:

  • Peptide competition assays

  • siRNA knockdown of ITGB1BP1

  • Comparison with alternative antibodies targeting different epitopes

It's worth noting that post-translational modifications may result in slight variations in the observed molecular weight. The search results indicate that ITGB1BP1 is a phosphoprotein, and its phosphorylation is regulated by cell-matrix interactions .

What methodological approaches can optimize ITGB1BP1 detection in tissues with low expression levels?

For tissues with low ITGB1BP1 expression, consider these optimization strategies:

For IHC applications:

• Antigen retrieval optimization: Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) to determine which provides better epitope accessibility

• Signal amplification: Implement tyramide signal amplification (TSA) or polymer-based detection systems

• Antibody concentration: Use higher antibody concentrations (e.g., 1:20 instead of 1:200) , but validate specificity at these concentrations

• Extended incubation: Increase primary antibody incubation times (overnight at 4°C)

• Reduce background: Use specialized blocking reagents containing both protein blockers and peroxidase inhibitors

For Western blot:

• Protein enrichment: Perform subcellular fractionation to concentrate ITGB1BP1

• Loading higher amounts: Increase protein loading (50-100 μg instead of standard 20-30 μg)

• Alternative visualization: Use chemiluminescent substrates with extended exposure times

• Sample preparation: Optimize lysis buffers to ensure complete extraction of ITGB1BP1

For immunofluorescence:

• Confocal microscopy: Use z-stacking to capture the full signal throughout the sample

• Fluorophore selection: Choose brighter fluorophores with minimal photobleaching

• Automated image analysis: Implement computational approaches to enhance signal detection and quantification

How can researchers effectively use ITGB1BP1 antibodies to investigate its role in integrin signaling pathways?

To investigate ITGB1BP1's role in integrin signaling pathways:

• Co-immunoprecipitation studies:

  • Use ITGB1BP1 antibodies to pull down protein complexes (0.5-4.0 μg antibody for 1.0-3.0 mg total protein)

  • Analyze co-precipitated proteins to identify binding partners, particularly integrin beta 1

  • Compare results between cells in suspension and those adhering to extracellular matrix proteins

• Proximity ligation assays:

  • Utilize ITGB1BP1 antibodies in combination with antibodies against integrins (particularly ITGB1)

  • Visualize and quantify direct protein interactions at single-molecule resolution

• Immunofluorescence co-localization:

  • Perform dual staining with ITGB1BP1 antibodies (1:200-1:800 dilution) and antibodies against focal adhesion proteins

  • Analyze co-localization at different stages of adhesion formation and maturation

  • Track changes during cell spreading, migration, and in response to extracellular matrix components

• Functional assays:

  • Combine antibody-based detection with ITGB1BP1 knockdown/overexpression

  • Measure downstream signaling events including:

    • Rho GTPase activation (CDC42 and RAC1)

    • AKT and ERK1/2 phosphorylation

    • Focal adhesion dynamics

    • Cell migration rates on different substrates (fibronectin, collagen, laminin)

• Quantitative analysis:

  • Use antibodies for quantifying ITGB1BP1 expression levels in different cell types

  • Correlate expression with integrin activation status and signaling output

What approaches can resolve conflicting data when using different ITGB1BP1 antibodies in experimental systems?

When facing conflicting results with different ITGB1BP1 antibodies:

• Epitope mapping analysis:

  • Compare immunogen information across antibodies

  • Antibodies targeting different regions of ITGB1BP1 may give different results due to:

    • Epitope accessibility in different experimental conditions

    • Post-translational modifications masking specific epitopes

    • Protein interactions blocking certain epitopes

• Validation using genetic approaches:

  • Test antibodies in ITGB1BP1 knockout systems

  • Perform siRNA/shRNA knockdown validation

  • Use overexpression systems with tagged ITGB1BP1 variants

• Cross-validation with multiple techniques:

  • If an antibody works in Western blot but not IHC, consider:

    • Fixation effects on epitope structure

    • Protein denaturation differences between techniques

    • Subcellular localization issues

• Isoform-specific detection:

  • ITGB1BP1 has multiple isoforms from alternatively spliced transcripts

  • Determine if antibodies recognize all or specific isoforms

  • The shorter form of ITGB1BP1 does not interact with beta1 integrin cytoplasmic domain

• Standardization of experimental conditions:

  • Use consistent sample preparation protocols

  • Standardize blocking reagents and antibody dilutions

  • Implement positive and negative controls across experiments

• Multi-antibody consensus approach:

  • Use at least three different antibodies targeting distinct epitopes

  • Consider results valid only when supported by majority of antibodies

  • Weight evidence based on validated antibody performance

How can ITGB1BP1 antibodies be utilized to investigate its role in angiogenesis and potential therapeutic implications?

ITGB1BP1 acts as a negative regulator of angiogenesis, making it an interesting target for therapeutic development. Here's how to investigate this role:

• Endothelial cell studies:

  • Use immunofluorescence (1:200-1:800 dilution) to track ITGB1BP1 localization during:

    • Tubule formation assays

    • Endothelial cell migration

    • Sprouting angiogenesis models

  • Correlate ITGB1BP1 expression/localization with:

    • AKT phosphorylation (which ITGB1BP1 promotes)

    • ERK1/2 phosphorylation (which ITGB1BP1 inhibits)

    • Notch signaling pathway activation

• In vivo angiogenesis models:

  • Implement IHC (1:20-1:200 dilution) for ITGB1BP1 in:

    • Developmental angiogenesis models

    • Tumor angiogenesis models

    • Ischemia-induced angiogenesis

  • Correlate ITGB1BP1 levels with vessel density, maturation, and functionality

• Mechanistic studies:

  • Use co-immunoprecipitation to identify ITGB1BP1 binding partners in endothelial cells

  • Investigate ITGB1BP1's effect on:

    • Endothelial cell proliferation markers

    • Integrin activation states using conformation-specific antibodies

    • Rho GTPase activation (particularly CDC42 and RAC1)

    • Focal adhesion dynamics during endothelial cell migration

• Therapeutic targeting validation:

  • Develop immunoassays to monitor ITGB1BP1 levels/modification state during:

    • Anti-angiogenic therapy response

    • Pro-angiogenic interventions

  • Assess correlation between ITGB1BP1 expression and:

    • Blood vessel normalization

    • Response to anti-VEGF therapies

    • Hypoxia and oxidative stress markers

• Pathway integration analysis:

  • Use antibodies to study how ITGB1BP1 intersects with:

    • VEGF signaling

    • Notch pathway components

    • ECM-integrin interactions

    • Mechanotransduction pathways

What considerations are important when selecting ITGB1BP1 antibodies for studying bone development and mineralization?

ITGB1BP1 plays crucial roles in bone mineralization and development. When selecting antibodies for these studies:

• Species specificity considerations:

  • Ensure antibody reactivity matches your experimental model (human, mouse, rat)

  • For evolutionary studies, consider antibodies that react across species

  • Validate species cross-reactivity experimentally rather than relying solely on sequence homology

• Developmental stage detection:

  • Verify antibody effectiveness across different osteoblast differentiation stages

  • Consider fixation methods that preserve both mineral and protein epitopes

  • Test antibodies in both developing and mature bone samples

• Detection in mineralized matrices:

  • Select antibodies validated for IHC in calcified tissues

  • Consider tissue processing methods:

    • Decalcification may affect epitope accessibility

    • Plastic embedding vs. paraffin embedding

    • Cryosectioning of undecalcified samples

• Functional domain targeting:

  • For mechanism studies, select antibodies targeting functional domains involved in:

    • Integrin binding

    • Rho GTPase regulation

    • Fibrillar adhesion formation

• Application-specific selection:

  • For tracking focal/fibrillar adhesion dynamics: IF-validated antibodies (1:200-1:800)

  • For protein-protein interactions: IP-validated antibodies (0.5-4.0 μg per reaction)

  • For expression level quantification: WB-validated antibodies (1:200-1:1000)

• Technical optimization for bone research:

  • Antigen retrieval optimization for mineralized tissues

  • Background reduction strategies for autofluorescent bone samples

  • Co-staining compatibility with bone markers

What methodological approaches can overcome challenges in using ITGB1BP1 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with ITGB1BP1 antibodies presents unique challenges due to its role in protein complexes. Here are methodological approaches to overcome these challenges:

• Optimized lysis conditions:

  • Use lysis buffers that maintain protein-protein interactions:

    • NP-40 or Triton X-100 (0.5-1%) for milder extraction

    • Avoid harsh detergents like SDS

  • Include phosphatase inhibitors to preserve ITGB1BP1's phosphorylation state

  • Add protease inhibitors to prevent degradation

• Crosslinking considerations:

  • For transient interactions, implement reversible crosslinking:

    • DSP (dithiobis(succinimidyl propionate)) at 1-2 mM

    • Formaldehyde at 0.1-1%

  • Optimize crosslinking time to capture interactions without excessive aggregation

• Antibody selection and strategy:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Consider the direction of the Co-IP:

    • IP with ITGB1BP1 antibody then blot for interaction partners

    • IP with partner antibodies then blot for ITGB1BP1

  • Compare results with different ITGB1BP1 antibodies targeting distinct epitopes

• Detection optimization:

  • Use antibodies validated for Western blot (1:200-1:1000)

  • Consider whether denaturation affects epitope recognition

  • Implement clean blot detection systems to reduce heavy chain interference

• Controls and validation:

  • Include isotype-matched IgG controls

  • Perform reciprocal Co-IPs when possible

  • Include known interaction controls:

    • ITGB1BP1 interaction with ITGB1 cytoplasmic domain

    • ITGB1BP1 interaction with Rho GTPases (CDC42, RAC1)

• Specific interaction enrichment:

  • Perform Co-IPs in specific cellular contexts:

    • Cells in suspension vs. adhered to ECM

    • During cell spreading/migration

    • After stimulation of specific signaling pathways

  • Subcellular fractionation before Co-IP to enrich relevant compartments

• Analysis of interaction dynamics:

  • Time-course studies following adhesion to different substrates

  • Comparison of interactions in different cell types (e.g., osteoblasts vs. endothelial cells)

  • Analysis of how phosphorylation affects interaction patterns