ITGB5 Antibody, HRP conjugated
Description
Introduction
Integrin beta-5 (ITGB5) is a transmembrane glycoprotein that forms heterodimers with integrin alpha-V (ITGAV) to mediate cell adhesion and signaling. The ITGB5 antibody conjugated with Horseradish Peroxidase (HRP) is a widely used research tool for detecting ITGB5 expression in biological samples. This review synthesizes data from diverse sources to provide a comprehensive analysis of its structure, applications, and research implications.
Structure and Function
The ITGB5 antibody is typically raised against specific epitopes within the ITGB5 protein. For example, the Abbexa HRP-conjugated antibody targets the 431–637 amino acid region , while the Cepham antibody recognizes a similar domain . HRP conjugation enables enzymatic amplification, making this antibody ideal for enzyme-linked immunosorbent assays (ELISA) and immunoblotting (WB).
| Supplier | Antibody Type | Immunogen | Applications |
|---|---|---|---|
| Abbexa | Rabbit Polyclonal | 431–637 AA | ELISA |
| Cepham | Rabbit Polyclonal | 431–637 AA | ELISA |
| BosterBio | Mouse Monoclonal | N/A | IHC, Flow Cytometry |
| Abcam | Goat Polyclonal | 750–C-terminus | WB |
Applications in Research
The ITGB5 antibody is primarily used in:
Research Findings: ITGB5 in Disease Pathogenesis
ITGB5 has been implicated in multiple cancers:
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Hepatocellular Carcinoma (HCC): Overexpression correlates with metastasis and poor prognosis via WNT/β-catenin signaling .
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Colorectal Cancer (CRC): High ITGB5 levels associate with TGF-β/EMT pathways and advanced TNM stages .
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Intrahepatic Cholangiocarcinoma (ICC): ITGB5 promotes proliferation and invasion, serving as a prognostic marker .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Elevated ITGB5 expression has been linked to cervical cancer. PMID: 26758421
- FAK activity regulates beta5 integrin expression and anchorage-independent cell growth in ovarian carcinoma. PMID: 24899686
- FRMD5 regulates tumor cell motility through a dual pathway involving its binding to integrin beta5 tail and ROCK1. PMID: 25448675
- Immunohistochemical analysis for alphavbeta3, alphavbeta5, and alphavbeta6 integrin subunits has been conducted and correlated with Ki67 and hypoxia-inducible factor (HIF)-1alpha indexes. PMID: 25150423
- Myo7a interacts with integrin beta5 and selectively promotes integrin alphavbeta5-mediated cell migration. PMID: 24997346
- Blocking experiments have shown that aV b5 and a6 significantly promoted hESC attachment in a 2% oxygen environment, while blocking CD44 inhibited cell attachment in 21% oxygen. PMID: 24455718
- Brain metastases ITGB5 expression exhibits significant heterogeneity based on tumor origin. PMID: 24294359
- Integrin-beta5 and zyxin mediate the formation of ventral stress fibers in response to transforming growth factor beta. PMID: 24036928
- A molecular model of integrin b5 structure has been prepared and its quality assessed. Integrin b5 active sites have been identified using in silico analysis tools. PMID: 23962022
- BDNF enhances the migration of chondrosarcoma by increasing beta5 integrin expression through a signaling pathway involving the TrkB receptor, PI3K, Akt, and NF-kappaB. PMID: 23874483
- The top primary GWAS association of Airway hyperresponsiveness was found in rs848788 (P-value 7.2E-07) located within the ITGB5 genes. PMID: 23984888
- Depletion of integrin beta5 in triple-negative breast carcinoma cells significantly reduced tumor take, growth, and angiogenesis, while reexpression of integrin beta5 restored this phenotype. These findings highlight the crucial role of integrin beta5 in the tumorigenic potential of breast carcinoma cells. PMID: 22824793
- High beta5-integrin protein expression has been associated with aggressive behavior in gastric cancer. PMID: 22561002
- Research suggests that the loss of integrin beta5 in polyomavirus-positive Merkel cell carcinoma may influence cell adhesion and migration. PMID: 22503669
- Cysteine-rich protein Cyr61 activates interleukin (IL)-6 production via the alphavbeta5/Akt/NF-kappaB signaling pathway in rheumatoid arthritis. PMID: 22547695
- Beta5-integrin adhesions contribute to TGFbeta-induced EMT and the tumorigenic potential of carcinoma cells. PMID: 20404485
- p21-activated kinase 4 phosphorylation of integrin beta5 Ser-759 and Ser-762 regulates cell migration. PMID: 20507994
- Src kinase-mediated activation of STAT3 and subsequent angiogenic gene expression mediate the effects of integrin alpha v beta 5 and may be exploited to enhance the paracrine activities of circulating angiogenic cells. PMID: 20431064
- Overexpression of the beta5 integrin in hematopoietic cells has been associated with the inhibition of cell proliferation and apoptosis. PMID: 11877043
- Studies have identified two distinct beta5 integrins: one with repeated-FNK (FNKFNK764-769) and another with single-FNK (FNK764-766) amino acid sequences in the cytoplasmic domain. PMID: 15979906
- Research suggests that the phagocytic function of beta5 integrin is regulated by an unconventional NPxY-talin-independent activation signal, indicating the existence of molecular switches in the beta5 cytoplasmic tail for adhesion and phagocytosis. PMID: 17963729
Q&A
How can researchers validate the specificity of an HRP-conjugated ITGB5 antibody in ELISA?
Methodological Answer:
To validate specificity, perform parallel assays using:
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Positive controls: Recombinant Human Integrin β5 protein (431-637AA) .
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Negative controls: Cell lysates from ITGB5-knockdown models (e.g., SW480/RKO CRC cells treated with siRNA ).
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Cross-reactivity checks: Test against related integrin subunits (e.g., ITGB1, ITGB3) using Western blot.
Expected Outcomes:
| Control Type | Expected Signal (OD450nm) | Interpretation |
|---|---|---|
| Recombinant ITGB5 | >2.0 | Validates antibody-epitope binding |
| ITGB5-knockdown lysate | <0.5 | Confirms target specificity |
| Unrelated integrin lysate | <0.3 | Rules out cross-reactivity |
Cite validation data from the antibody datasheet (e.g., >95% purity via Protein G purification ) and independent studies (e.g., siRNA-mediated ITGB5 suppression in CRC ).
What storage conditions preserve HRP conjugate activity for long-term use?
Methodological Answer:
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Temperature: Aliquot and store at -20°C; avoid repeated freeze-thaw cycles .
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Light exposure: Shield from direct light using amber tubes.
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Buffer composition: Maintain in 0.01 M PBS (pH 7.4) with 50% glycerol to prevent aggregation .
Stability Testing:
| Condition | Activity Retention (6 months) |
|---|---|
| -20°C, protected | 95–100% |
| 4°C, unprotected | <50% |
| Room temperature, 1 week | <10% |
How is the optimal antibody dilution determined for immunohistochemistry (IHC)?
Methodological Answer:
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Perform a dilution series (1:100 to 1:1000) on FFPE CRC tissues with known ITGB5 expression levels .
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Use signal-to-noise ratios (SNR) to identify the dilution with maximal specific staining and minimal background.
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Validate with a blocking peptide (e.g., immunogen fragment 431-637AA) to confirm signal loss at optimal dilution.
Example Data:
| Dilution | SNR (Tumor vs. Stroma) | Background Intensity |
|---|---|---|
| 1:200 | 8.5 | Low |
| 1:500 | 6.2 | Minimal |
| 1:1000 | 3.1 | Undetectable |
How can ITGB5 antibody be integrated into multiplex assays to study tumor-immune interactions?
Methodological Answer:
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Multiplex immunofluorescence: Combine ITGB5-HRP with antibodies against CD68 (macrophages), CD8 (T cells), and Pan-CK (epithelial cells). Use tyramide signal amplification (TSA) to avoid spectral overlap.
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Image analysis: Quantify ITGB5 expression in regions of interest (ROIs) colocalizing with immune markers using software like HALO or QuPath.
Case Study:
In CRC tissues, ITGB5-high regions show 2.3-fold greater M2 macrophage infiltration (ρ = 0.67, p < 0.01) .
What methods confirm ITGB5’s role in Wnt/β-catenin signaling using this antibody?
Methodological Answer:
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Co-staining: Perform IHC for β-catenin (nuclear) and ITGB5 (membrane/cytoplasmic) on serial CRC sections.
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Functional assays: Treat ITGB5-knockdown cells with Wnt3a and measure β-catenin levels via Western blot .
Key Findings:
| Condition | β-Catenin (Nuclear) | ITGB5 Expression |
|---|---|---|
| Control | High | High |
| ITGB5 siRNA | Reduced by 64% | Low |
| Wnt3a + ITGB5 siRNA | Partial restoration (38%) | Low |
How should discrepancies in ITGB5 expression data across CRC studies be resolved?
Methodological Answer:
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Meta-analysis: Harmonize data using R packages (e.g.,
metafor) to assess heterogeneity across datasets (e.g., GSE39582 vs. GSE17538 ). -
Subgroup analysis: Stratify by clinicopathological variables (e.g., TNM stage, metastasis status) where ITGB5 correlates with advanced disease (χ² p = 0.045) .
Contradiction Resolution Framework:
| Step | Action | Example |
|---|---|---|
| 1 | Re-analyze raw data | Normalize microarray data using RMA |
| 2 | Validate with orthogonal methods | Compare IHC and RNA-seq results |
| 3 | Control for covariates | Adjust for tumor purity or stromal content |
Synthesis of Key Findings
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Clinical Relevance: High ITGB5 expression correlates with advanced TNM stage (OR = 2.1, p = 0.045), lymph node metastasis (OR = 1.8, p = 0.036), and poor survival (HR = 1.62, p = 0.003) .
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Mechanistic Insights: ITGB5 upregulates Wnt/β-catenin targets (e.g., AXIN2, LEF1) and EMT markers (e.g., SNAI1, VIM) .
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Technical Considerations: Antibody specificity is critical due to homology with integrin β subunits (e.g., 43% sequence similarity with ITGB1).
