ITGB5 Antibody

What is the molecular weight of ITGB5 and why do I observe different band sizes in Western blot?

ITGB5 has a theoretical molecular weight of approximately 88 kDa, but researchers often observe variability in band sizes:

• 100 kDa in HT-29 human colon adenocarcinoma cells under reducing conditions

• 129 kDa in MG-63 human osteosarcoma cell line using Simple Western analysis

This variability stems from:

• Post-translational modifications, particularly glycosylation

• Cell type-specific processing mechanisms

• Differences in sample preparation and electrophoresis conditions

To confirm band identity, always include positive controls such as MG-63 or HT-29 cell lysates. Consider deglycosylation experiments to verify the contribution of glycosylation to the observed molecular weight.

Which cell lines serve as reliable positive controls for ITGB5 antibody validation?

Based on published literature and commercial validation data, these cell lines consistently express ITGB5 and serve as excellent positive controls:

For negative controls, ITGB5 knockdown using siRNA in these cell lines provides the most definitive validation of antibody specificity.

What are the optimal protocols for detecting ITGB5 by immunocytochemistry and immunofluorescence?

For successful ITGB5 immunostaining:

Sample Preparation:

• For adherent cells: Grow on collagen-coated coverslips (enhances ITGB5 expression)

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 for 10 minutes (for intracellular domains)

Immunostaining Protocol:

• Block with 5-10% normal serum (matching secondary antibody host) for 1 hour

• Incubate with ITGB5 primary antibody (5-10 μg/ml) for 3 hours at room temperature or overnight at 4°C

• Wash 3× with PBS

• Apply appropriate fluorophore-conjugated secondary antibody (e.g., NorthernLights™ 557-conjugated Anti-Sheep IgG)

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Critical Optimization Steps:

• Antibody concentration: Titrate between 2-10 μg/ml (5 μg/ml is a good starting point)

• Include positive control cells (MG-63 shows specific cytoplasmic staining)

• For co-localization studies, consider dual staining with αV antibodies to detect the heterodimer

How can I optimize Western blot protocols for consistent ITGB5 detection?

For reproducible Western blot results with ITGB5 antibodies:

Sample Preparation:

• Lyse cells in RIPA buffer with protease inhibitors

• Load 20-50 μg protein per lane

• Use reducing conditions for consistent results

Optimized Protocol:

• Separate proteins on 8-10% SDS-PAGE (ITGB5 is relatively large)

• Transfer to PVDF membrane (recommended over nitrocellulose for ITGB5)

• Block with 5% non-fat milk or BSA in TBST for 1 hour

• Incubate with ITGB5 primary antibody (0.1-1 μg/ml) overnight at 4°C

• Wash 3-5× with TBST

• Incubate with appropriate HRP-conjugated secondary antibody (1:2000-1:5000)

• Detect using enhanced chemiluminescence

Troubleshooting Tips:

• Use Western Blot Buffer Group 1 for optimal results with ITGB5

• Expected molecular weight varies (88-129 kDa) depending on cell type

• Include positive controls like MG-63 or HT-29 cell lysates

• For weak signals, consider longer exposure times or signal amplification systems

How should ITGB5 antibodies be stored and handled to maintain activity?

Proper storage and handling are critical for maintaining antibody performance:

Short-term storage (up to two weeks):

• Store at 4°C

• Avoid repeated freeze-thaw cycles

Long-term storage:

• Divide into small aliquots (≥20 μl) to minimize freeze-thaw cycles

• Store at -20°C to -70°C

• For concentrate products, adding equal volume of glycerol as cryoprotectant before freezing is recommended

Handling recommendations:

• Most ITGB5 antibodies remain stable for approximately 12 months from receipt when stored properly at -20°C to -70°C

• Thaw aliquots completely before use and mix gently

• Return to appropriate storage condition immediately after use

• Never store diluted antibody for extended periods

The shelf-life at 4°C is highly variable between antibody preparations, so for long-term applications, freezing is strongly recommended .

How can ITGB5 antibodies be used to study cancer progression mechanisms?

ITGB5 has emerged as an important biomarker and functional mediator in cancer progression, particularly in gastric cancer and glioblastoma:

Expression Analysis in Tumors:

• IHC/IF: Compare ITGB5 levels between tumor and adjacent normal tissues

• Western blot: Quantify ITGB5 protein levels across cancer stages

• qPCR: Measure ITGB5 mRNA expression in conjunction with protein studies

Functional Studies:

• RNA interference: siRNA knockdown of ITGB5 has been shown to suppress proliferation and migration of gastric cancer cell lines

• Function-blocking antibodies: Use blocking antibodies (e.g., IPI-ITGAV/ITGB5.9) to inhibit ITGB5 function

• Migration/invasion assays:

  • Transwell migration: Seed cells at 2×10⁴ cells/200μl in upper chamber with 0.2% FBS

  • Invasion assay: Coat chambers with 500 ng/ml Matrigel before seeding 4×10⁴ cells/200μl

Mechanistic Investigations:

• Focus on PI3K-Akt, ECM-receptor interaction, and TGF-beta pathways, which are linked to ITGB5 function

• Assess relationship with CD276, as ITGB5 knockdown results in decreased CD276 expression in gastric cancer

• Investigate correlation with EMT markers, as ITGB5 silencing has been shown to decrease their expression

Research demonstrates that elevated ITGB5 expression correlates with poor prognosis in both gastric cancer and glioblastoma, highlighting its potential as both a prognostic marker and therapeutic target .

What methods can be used to investigate ITGB5's role in immune cell infiltration?

ITGB5 expression is significantly correlated with immune cell infiltration in tumors, particularly macrophages:

Computational Analysis:

• Use TIMER algorithm to assess correlation between ITGB5 expression and tumor-infiltrating immune cells

• Apply CIBERSORT to calculate relative proportions of 22 immune cell types based on ITGB5 expression

• Employ TISIDB database to examine relationships between ITGB5 and immunoregulatory genes

Experimental Approaches:

• Multiplex immunohistochemistry: Co-stain ITGB5 with immune markers (CD4, CD8, CD68)

• Flow cytometry: Analyze correlation between ITGB5 expression and immune cell populations

• Functional co-culture assays: Assess how ITGB5-expressing tumor cells influence immune cell behavior

Key Research Findings:

• ITGB5 expression positively correlates with infiltration of CD4+ T cells (cor = 0.155, p = 2.91e-03), macrophages (cor = 0.314, p = 6.51e-10), and dendritic cells (cor = 0.132, p = 1.06e-02)

• The macrophage infiltration significantly correlates with prognosis of gastric cancer patients

• ITGB5 expression is significantly correlated with macrophage markers, including M1 markers (NOS2, IL1B, CD86), M2 markers (CSF1R, MRC1, CD163), and tumor-associated macrophage markers (MARCO, CSF1R, CD40)

These findings suggest ITGB5 might play a crucial role in the immune microenvironment of tumors, potentially through promoting M2 macrophage polarization and inhibiting antitumor immunity .

How can researchers use ITGB5 antibodies to investigate angiogenesis mechanisms?

ITGB5 is implicated in angiogenesis, particularly in areas of microvascular proliferation in glioblastoma:

In vitro Angiogenesis Assays:

• Tube formation assay: Plate endothelial cells on Matrigel with ITGB5 function-blocking antibodies

• Endothelial cell migration: Assess migration with and without ITGB5 antibody blockade

• Co-culture systems: Combine endothelial cells with ITGB5-expressing tumor cells

Tissue Analysis Approaches:

• Immunohistochemistry: Co-stain for ITGB5 and endothelial markers (CD31, CD34)

• Comparative analysis: Examine ITGB5 expression in areas of microvascular proliferation versus other tumor regions

Significant Research Findings:

• Data from the Ivy database shows ITGB5 is overexpressed in areas of microvascular proliferation relative to other regions of glioblastoma tumors

• ITGB5 is more highly expressed in IDH1-wild-type compared to IDH1-mutant glioblastoma, correlating with the more aggressive vascular phenotype of wild-type tumors

Function-blocking antibodies that target the αVβ5 heterodimer (like IPI-ITGAV/ITGB5.9) can be valuable tools to elucidate the specific contribution of this integrin to pathological blood vessel formation .

How can ITGB5 serve as a prognostic biomarker in cancer research?

ITGB5 has demonstrated significant value as a prognostic biomarker in multiple cancer types:

Gastric Cancer:

Glioblastoma:

Methodological Approaches:

• IHC scoring systems (0-3+ or H-score) for ITGB5 protein expression

• Kaplan-Meier survival analysis with log-rank test to assess prognostic significance

• Multivariate Cox regression to evaluate independence from other prognostic factors

• Combined assessment with other biomarkers (e.g., CEA and CA19-9 in gastric cancer) to improve diagnostic accuracy

These findings suggest ITGB5 could be incorporated into clinical risk stratification systems to identify patients with more aggressive disease who might benefit from intensified treatment regimens.

What are the emerging clinical applications of ITGB5 as a circulating biomarker?

Recent research has identified ITGB5 as a promising circulating biomarker, particularly in gastric cancer:

Serum ITGB5 as a Diagnostic Marker:

• ELISA measurements show significantly elevated serum ITGB5 levels in gastric cancer patients compared to healthy controls

• The combined assessment of ITGB5, CEA, and CA19-9 improved diagnostic accuracy compared to individual markers

Potential Clinical Applications:

• Early detection of cancer in high-risk populations

• Monitoring treatment response and disease recurrence

• Complementing tissue-based markers for comprehensive assessment

• Patient stratification for clinical trials

Methodological Considerations:

• Standardized ELISA protocols are essential for reliable quantification

• Pre-analytical variables (sample collection, processing, storage) must be controlled

• Reference ranges need to be established in diverse populations

• Validation in prospective clinical studies is required before routine implementation

Extracellular Vesicle Analysis:

• vFC™ (vesicle flow cytometry) with vTAG™ anti-human ITGB5 antibody enables detection of ITGB5 on extracellular vesicles with a limit of detection as low as 30 molecules per vesicle

• This emerging technology may provide additional information on tumor-derived vesicles in liquid biopsies

While promising, further prospective studies with larger cohorts are needed to fully establish the clinical utility of circulating ITGB5 as a cancer biomarker.

What controls should be included in ITGB5 antibody experiments to ensure validity?

A comprehensive control strategy for ITGB5 antibody experiments should include:

Positive Controls:

• Cell lines: MG-63 (osteosarcoma), HT-29 (colon adenocarcinoma), MDA-MB-231 (breast cancer)

• Tissues: Gastric cancer or glioblastoma specimens (particularly mesenchymal subtype GBM)

• Recombinant ITGB5 protein (for Western blot or ELISA)

Negative Controls:

• Primary antibody omission: Reveals non-specific binding of secondary antibody

• Isotype control: Matches primary antibody's host species and isotype

• siRNA knockdown: Cells treated with ITGB5-targeting siRNA should show reduced signal

• Normal tissues with low ITGB5 expression

Technical Controls:

• Loading control: Use housekeeping proteins (β-actin, GAPDH) for Western blot normalization

• Tissue integrity control: H&E staining of adjacent sections in IHC

• Cell viability marker: Ensures observed patterns aren't due to cell death

Validation Across Methods:

• Correlate protein detection with mRNA expression (qPCR)

• Confirm findings with multiple antibody clones targeting different epitopes

• For heterodimer studies, co-stain with alpha V (ITGAV) antibodies

For flow cytometry specifically, include unstained, single-stained, and FMO (fluorescence minus one) controls, and use viability dye to exclude dead cells from analysis.

How can researchers address non-specific staining issues with ITGB5 antibodies?

To minimize background and optimize ITGB5 immunostaining:

Antibody Optimization:

• Titrate antibody concentrations (start with 2-5 μg/ml for IHC/IF)

• Consider different antibody formats (monoclonal vs. polyclonal)

• Reduce incubation time or temperature

• Try different antibody clones if persistent issues occur

Blocking Improvements:

• Extend blocking time (60+ minutes)

• Test different blockers (BSA, normal serum, commercial blockers)

• Add specific blocking steps for endogenous biotin or peroxidase

• Include Fc receptor blocking when staining tissues with high immune cell content

Protocol Modifications:

• Increase number and duration of wash steps

• Optimize detergent concentration in wash buffers

• Filter antibody dilutions to remove aggregates

• Consider using commercial antibody diluents designed to reduce background

Sample-Specific Approaches:

• Optimize antigen retrieval method (citrate pH 6.0 vs. EDTA pH 9.0)

• Adjust fixation protocol (duration, fixative type)

• For fluorescence, use autofluorescence quenching reagents

• Counterstain optimization for better contrast

Researchers have successfully used NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody for specific ITGB5 detection in MG-63 human osteosarcoma cell line with minimal background .