ITGB4 (Ab-1510) Antibody

What is ITGB4 (Ab-1510) Antibody and what specific epitope does it recognize?

ITGB4 (Ab-1510) Antibody is a rabbit polyclonal antibody that detects endogenous levels of total ITGB4 protein. This antibody is specifically designed to recognize a synthetic non-phosphopeptide derived from human ITGB4 around the phosphorylation site of tyrosine 1510 (R-D-Y-S-T) . The antibody was affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography, ensuring high specificity . ITGB4 is alternatively known as CD104 antigen, GP150, ITB4, integrin beta-4, or integrin beta-4C, and has an SDS-PAGE molecular weight of approximately 202 kDa .

What are the validated research applications for ITGB4 (Ab-1510) Antibody?

ITGB4 (Ab-1510) Antibody has been validated for multiple research applications:

• Western Blotting (WB): Effective at dilutions of 1:1,000-1:2,000

• Immunohistochemistry (IHC): Recommended dilutions range from 1:50-1:200

The antibody shows specific reactivity to human samples, with some commercially available variants also exhibiting reactivity to mouse samples . Validation has been performed across multiple cell lines including A549, Jurkat, and BxPC-3 cells, confirming specificity through the observation of the expected molecular weight band (approximately 200-210 kDa) .

How should ITGB4 (Ab-1510) Antibody be stored and handled for optimal experimental results?

For optimal performance and longevity, ITGB4 (Ab-1510) Antibody should be stored at -20°C . The antibody is typically provided in a formulation of rabbit IgG in phosphate-buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol . This formulation helps maintain antibody stability during storage.

When handling the antibody:

• Avoid repeated freeze-thaw cycles

• Aliquot the antibody upon first thaw if multiple experiments are planned

• Allow the antibody to equilibrate to room temperature before opening the vial

• Return to -20°C immediately after use

• When diluting, use fresh, cold buffer solutions

What is the recommended Western blotting protocol for optimal results with ITGB4 (Ab-1510) Antibody?

The following Western blotting protocol has been validated for ITGB4 (Ab-1510) Antibody:

Sample Preparation and SDS-PAGE:

• Prepare cell or tissue lysates using standard lysis buffers

• Load 10-40 μg of protein per lane (10 μg shown effective for BxPC-3, SW480, and pancreatic tissue samples)

• Separate proteins on an SDS-PAGE gel appropriate for high molecular weight proteins (ITGB4 is approximately 202-210 kDa)

Transfer and Blocking:

• Transfer proteins to a nitrocellulose membrane

• Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

Antibody Incubation:

• Dilute ITGB4 (Ab-1510) Antibody at 1:1,000 in blocking buffer

• Incubate overnight at 4°C

• Wash four times with TBST

• Incubate with HRP-conjugated secondary antibody (Anti-Rabbit IgG) at 1:1,000 dilution for 1 hour at room temperature

• Wash four times with TBST

Detection:

• Apply ECL substrate and expose

• Exposure time of approximately 30 seconds has been effective for standard samples

The predicted band size is 202 kDa, but the observed band typically appears at approximately 210 kDa in most cell lines .

What controls are essential when using ITGB4 (Ab-1510) Antibody in experimental procedures?

When designing experiments with ITGB4 (Ab-1510) Antibody, the following controls are critical:

Positive Controls:

• A431 and A549 cell lines have been validated as positive controls for ITGB4 expression

• BxPC-3 (human pancreatic adenocarcinoma cells) and SW480 (human colon adenocarcinoma) cell lysates consistently show robust ITGB4 expression

• Human pancreas tissue lysate is an effective positive control for tissue samples

Negative Controls:

• ITGB4 knockout cell lines (such as ITGB4 knockout A549 cells) serve as excellent negative controls to confirm antibody specificity

• Primary antibody omission control to assess non-specific binding of secondary antibody

• Isotype control (rabbit IgG) to evaluate non-specific binding

Loading Controls:

• Calnexin (CANX) has been validated as an appropriate loading control when conducting Western blot with ITGB4 antibodies

Additional Validation:

• Using siRNA knockdown of ITGB4 (siITGB4) as demonstrated in the literature can further validate antibody specificity in functional studies

How can ITGB4 (Ab-1510) Antibody be optimized for immunohistochemistry applications?

For optimal immunohistochemistry results with ITGB4 (Ab-1510) Antibody:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin

• Process and embed in paraffin

• Section at 4-6 μm thickness

Antigen Retrieval:

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Heat at 95-98°C for 15-20 minutes, then cool to room temperature

Staining Protocol:

• Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

• Apply protein blocking solution for 30 minutes

• Dilute ITGB4 (Ab-1510) Antibody at 1:50-1:200 in antibody diluent

• Incubate sections overnight at 4°C or 60 minutes at room temperature

• Wash thoroughly with PBS or TBS

• Apply appropriate HRP-conjugated secondary antibody

• Develop with DAB substrate

• Counterstain with hematoxylin

Tissue Considerations:

• Human breast cancer tissue samples have been validated to show clear membrane and cytoplasmic staining for ITGB4

• Expression patterns should be evaluated in context of tissue architecture, particularly noting epithelial cell boundaries where ITGB4 is expected to localize in hemidesmosomes

How can ITGB4 (Ab-1510) Antibody be utilized to investigate ITGB4's role in cancer progression?

ITGB4 has been identified as a significant oncogenic factor in multiple cancer types. ITGB4 (Ab-1510) Antibody can be employed in several advanced research applications to study its role in cancer:

Expression Analysis Across Cancer Types:
Research has revealed that ITGB4 expression levels are significantly altered in multiple cancer types, including bladder urothelial carcinoma, cervical squamous cell carcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, glioblastoma, head and neck squamous cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, and pancreatic adenocarcinoma . ITGB4 (Ab-1510) Antibody can be used to:

• Compare ITGB4 expression between tumor and adjacent normal tissues

• Correlate expression levels with clinical parameters and patient outcomes

• Create tissue microarrays for high-throughput analysis across multiple patient samples

Prognostic Value Assessment:
ITGB4 expression correlates with prognosis in several cancers:

ITGB4 (Ab-1510) Antibody can help establish these correlations through immunohistochemical scoring of patient samples.

Functional Studies:
To investigate the mechanistic role of ITGB4, combine ITGB4 (Ab-1510) Antibody with genetic manipulation approaches:

• Use siRNA knockdown of ITGB4 (as demonstrated in lung adenocarcinoma cell lines) and confirm knockdown efficiency via Western blot with ITGB4 (Ab-1510) Antibody

• Assess phenotypic changes in:

  • Proliferation (using Cell Counting Kit-8 assays)

  • Colony formation

  • Apoptosis (using flow cytometry)

  • Migration (using wound healing assays)

  • Invasion (using Transwell assays)

What methodologies can be employed to study the phosphorylation status of ITGB4 at tyrosine 1510 and its impact on signaling?

Since ITGB4 (Ab-1510) Antibody specifically recognizes the region around the tyrosine 1510 phosphorylation site, it provides a valuable tool for studying this post-translational modification:

Phosphorylation Status Analysis:

• Use phosphatase inhibitors in lysis buffers when preparing samples to preserve phosphorylation status

• Compare results with phospho-specific antibodies that recognize only the phosphorylated form of tyrosine 1510

• Perform lambda phosphatase treatment on parallel samples to confirm phosphorylation-dependent effects

Signaling Pathway Investigation:
ITGB4 participates in multiple signaling cascades. To investigate these pathways:

• Stimulate cells with relevant growth factors known to interact with ITGB4, such as:

  • Neuregulin 1 (NRG1) - ITGA6:ITGB4 binds to NRG1 via its EGF domain

  • Insulin-like growth factor 1 (IGF1) - ITGB4 binding is essential for IGF1 signaling

  • Insulin-like growth factor 2 (IGF2) - ITGB4 binding is essential for IGF2 signaling

• Perform co-immunoprecipitation experiments:

  • Immunoprecipitate with ITGB4 (Ab-1510) Antibody

  • Probe for interacting partners (NRG1, IGF1, IGF2, ERBB receptors)

  • Investigate downstream signaling components

• Analyze phosphorylation-dependent protein interactions:

  • Compare wild-type ITGB4 with Y1510F mutants (tyrosine to phenylalanine)

  • Assess effects on binding partners and downstream signaling

What are the key considerations when designing ITGB4 knockdown experiments and analyzing downstream effects?

Based on published research using ITGB4 knockdown approaches, several methodological considerations are important:

Knockdown Strategy Selection:

• siRNA approach:

  • Multiple siRNAs targeting different regions of ITGB4 mRNA should be used to confirm specificity

  • siITGB4#1 and siITGB4#2 have been validated in A549 and PC9 lung adenocarcinoma cell lines

  • Validate knockdown efficiency via Western blot using ITGB4 (Ab-1510) Antibody

  • Typical knockdown efficiency should reduce expression by >70%

• shRNA approach for stable knockdown:

  • Consider lentiviral delivery systems for hard-to-transfect cells

  • Establish stable cell lines through antibiotic selection

  • Verify sustained knockdown over experimental timeframes

Experimental Design for Functional Studies:

Downstream Molecular Analysis:
After confirming ITGB4 knockdown:

• Perform transcriptomic analysis to identify differentially expressed genes

• Conduct pathway enrichment analysis:

  • KEGG pathway analysis has identified multiple altered pathways including cell cycle, p53 signaling, and ECM-receptor interaction pathways

  • Reactome pathway analysis provides complementary insights into affected biological processes

• Validate key altered genes with qRT-PCR and Western blot analysis

How does ITGB4 interact with other integrin subunits and extracellular matrix components in different tissue contexts?

ITGB4 primarily pairs with integrin α6 (ITGA6) to form the α6β4 heterodimer. This complex has distinctive functions across tissue types:

Epithelial Tissue Context:

• In normal epithelial cells, ITGB4 is a critical structural component of hemidesmosomes

• ITGB4 (Ab-1510) Antibody can be used to assess the localization of ITGB4 at the basal surface of epithelial cells where it interacts with basement membrane laminin

• Co-staining with hemidesmosome components (such as plectin, BP180, BP230) can reveal proper assembly or dysregulation in pathological conditions

Cancer Context:

• During cancer progression, ITGB4 often relocalizes from hemidesmosomes to leading edges of migrating cells

• This relocalization coincides with altered signaling functions:

  • Enhanced cell migration

  • Increased invasion

  • Altered growth factor signaling

Methodological Approach to Study Interactions:

• Co-immunoprecipitation:

  • Pull down with ITGB4 (Ab-1510) Antibody

  • Probe for associated proteins (ITGA6, laminin subunits, signaling molecules)

• Proximity Ligation Assay (PLA):

  • Detect in situ protein-protein interactions between ITGB4 and potential binding partners

  • Provides spatial information about where interactions occur within cells

• Immunofluorescence Co-localization:

  • Use ITGB4 (Ab-1510) Antibody in combination with antibodies against:

    • Extracellular matrix components (laminins, particularly laminin-332)

    • Signaling molecules (EGFR, ErbB2, c-Met)

    • Cytoskeletal proteins (actin, plectin)

  • Analyze co-localization coefficients in different cellular regions

• Tissue-Specific Expression Pattern Analysis:

  • Compare ITGB4 localization patterns across multiple tissue types using tissue microarrays

  • Correlate with pathological features and other molecular markers

What are common technical challenges when using ITGB4 (Ab-1510) Antibody and how can they be resolved?

When working with ITGB4 (Ab-1510) Antibody, researchers may encounter several technical challenges:

Western Blotting Challenges:

Immunohistochemistry Challenges:

Validation Strategies:

• Use ITGB4 knockout cell lines as negative controls (e.g., ITGB4 knockout A549 cells)

• Compare with alternative ITGB4 antibodies targeting different epitopes

• Perform peptide competition assays to confirm specificity

• Include siRNA knockdown samples as biological validation controls

How should researchers interpret ITGB4 expression data in relation to cancer prognosis and treatment response?

When interpreting ITGB4 expression data, several contextual factors must be considered:

Cancer Type-Specific Considerations:
ITGB4 expression has distinct prognostic implications across cancer types:

Multi-parameter Analysis Recommendations:

• Always perform multivariate analysis considering:

  • Tumor stage and grade

  • Patient demographic factors

  • Other molecular markers

  • Treatment history

• Quantify ITGB4 expression using standardized methods:

  • For IHC: Use H-score or Allred scoring system

  • For Western blot: Normalize to validated housekeeping proteins

  • For qRT-PCR: Use multiple reference genes for normalization

• Establish threshold values based on:

  • ROC curve analysis for outcome prediction

  • Population distribution (median, quartiles)

  • Biologically relevant expression levels from functional studies

Integration with Functional Data:
When interpreting ITGB4 expression data, consider the functional evidence showing that ITGB4 knockdown:

• Inhibits cell proliferation

• Promotes apoptosis

• Reduces migration and invasion capabilities

These functional effects provide mechanistic context for the prognostic associations observed in clinical samples.

What complementary techniques should be used alongside ITGB4 (Ab-1510) Antibody to fully characterize ITGB4's role in experimental models?

A comprehensive characterization of ITGB4's role requires integration of multiple techniques:

Genomic and Transcriptomic Approaches:

• RNA-Seq analysis after ITGB4 knockdown to identify differentially expressed genes

• ChIP-Seq to identify transcription factors regulating ITGB4 expression

• Single-cell RNA-Seq to characterize ITGB4 expression heterogeneity within tumor samples

Proteomic Approaches:

• Mass spectrometry to identify ITGB4 binding partners and post-translational modifications

• Reverse phase protein array (RPPA) to assess activation of multiple signaling pathways

• Co-immunoprecipitation followed by western blot to confirm specific protein interactions

Functional Genomics:

• CRISPR-Cas9 knockout of ITGB4 to create stable cell lines for long-term studies

• CRISPR activation or inhibition systems to modulate ITGB4 expression levels

• Site-directed mutagenesis of key phosphorylation sites (including Y1510) to assess functional significance

Advanced Imaging Techniques:

• Super-resolution microscopy to visualize ITGB4 in hemidesmosomes and other cellular structures

• Live-cell imaging with fluorescently tagged ITGB4 to monitor dynamics during cell migration

• FRET/FLIM analysis to detect protein-protein interactions in live cells

In Vivo Models:

• Patient-derived xenografts (PDX) with variable ITGB4 expression levels

• Genetically engineered mouse models with conditional ITGB4 knockout

• Orthotopic tumor models to assess metastatic potential in relation to ITGB4 expression

Data Integration Strategy:
When designing multi-omics experiments to study ITGB4:

• Establish clear temporal sequence for data collection

• Use consistent experimental conditions across platforms

• Develop computational pipelines to integrate diverse data types

• Validate key findings with orthogonal techniques

• Consider pathway-based analysis rather than focusing solely on individual genes/proteins

How might ITGB4 (Ab-1510) Antibody be utilized in studying the relationship between ITGB4 and treatment resistance in cancer?

Emerging research suggests ITGB4 may contribute to treatment resistance in multiple cancer types. ITGB4 (Ab-1510) Antibody can be applied to investigate this relationship through several experimental approaches:

Therapy Response Correlation Studies:

• Analyze ITGB4 expression in matched pre- and post-treatment tumor samples

• Correlate expression levels with:

  • Radiotherapy response

  • Chemotherapy resistance

  • Targeted therapy outcomes

  • Immunotherapy efficacy

Mechanistic Investigation:

• Create therapy-resistant cell lines and analyze ITGB4 expression changes

• Perform ITGB4 knockdown in resistant cells to assess resensitization

• Investigate ITGB4-dependent survival pathways activated during treatment

Combination Therapy Approaches:

• Test combination of ITGB4-targeting strategies with standard therapies

• Evaluate synergistic effects on cell death, migration, and invasion

• Analyze pathway alterations using phospho-protein arrays and transcriptomics

Biomarker Development:

• Develop standardized IHC protocols using ITGB4 (Ab-1510) Antibody for patient stratification

• Establish cutoff values predictive of therapy response

• Integrate with other molecular markers for improved predictive power

What are the key technical considerations when using ITGB4 (Ab-1510) Antibody in multi-parameter analyses such as multiplexed imaging or CyTOF?

As cancer research moves toward more comprehensive cellular analysis, ITGB4 (Ab-1510) Antibody can be incorporated into multiplexed techniques with the following considerations:

For Multiplexed Immunofluorescence:

• Antibody Validation:

  • Confirm specificity in single-marker controls

  • Test for cross-reactivity with other antibodies in the panel

  • Validate signal after multiple stripping/reprobing cycles

• Panel Design:

  • Select complementary markers (e.g., ITGA6, laminin, epithelial markers)

  • Choose fluorophores with minimal spectral overlap

  • Include proper controls for autofluorescence and non-specific binding

• Optimization Protocol:

  • Test antibody in decreasing dilutions (1:50, 1:100, 1:200, 1:500)

  • Optimize signal amplification requirements

  • Determine ideal fixation conditions that preserve multiple epitopes

For CyTOF/Mass Cytometry:

• Metal Conjugation:

  • Select metal isotope with minimal spillover to other channels

  • Validate antibody function after metal conjugation

  • Titrate to determine optimal concentration

• Sample Processing:

  • Optimize cell fixation to preserve ITGB4 epitope

  • Develop appropriate permeabilization protocol for internal epitopes

  • Include barcoding strategy for batch processing

• Data Analysis:

  • Apply dimensionality reduction techniques (t-SNE, UMAP)

  • Develop gating strategies incorporating ITGB4 expression

  • Correlate ITGB4 with other markers in identified cell clusters

For Spatial Transcriptomics Integration:

• Combine ITGB4 protein detection with mRNA analysis

• Correlate protein expression with spatial gene expression patterns

• Analyze tumor-stroma interface for ITGB4 expression patterns

Research Data Tables