ITGB1 Antibody

What is ITGB1 and what biological functions does it serve?

ITGB1 (Integrin beta 1) is a transmembrane protein involved in cell adhesion, migration, proliferation, differentiation, and signaling. It forms heterodimeric complexes with different alpha integrin subunits to create functional receptors for extracellular matrix (ECM) molecules such as fibronectin, laminin, and collagen. This protein plays critical roles in numerous physiological and pathological processes, including embryonic development, wound healing, immune response, and cancer metastasis. Dysregulation of ITGB1 signaling has been implicated in various diseases, including cardiovascular disease, inflammation, and cancer .

Also known as CD29, ITGB1 is a 130 kDa single chain type I glycoprotein broadly expressed on the majority of hematopoietic and non-hematopoietic cells, including leukocytes, platelets, fibroblasts, endothelial cells, epithelial cells, and mast cells .

What applications are ITGB1 antibodies validated for in research settings?

ITGB1 antibodies are validated for multiple research applications, with specific recommended dilutions varying by manufacturer and application:

Different ITGB1 antibodies may be optimized for specific applications. For example, some antibodies are specifically designed for function-blocking experiments , while others are conjugated with fluorescent markers like FITC for flow cytometry applications .

How can I validate the specificity of an ITGB1 antibody?

Validating ITGB1 antibody specificity requires multiple approaches:

• Knockout validation: Use ITGB1 knockout cell lines as negative controls. Western blot analysis shows that specific ITGB1 antibodies produce bands at 110-140 kDa in wild-type HeLa cell lysates while showing no signal in ITGB1 knockout cell lines .

• Molecular weight verification: Confirm that observed bands match the expected molecular weight range. While the predicted molecular weight of ITGB1 is 88 kDa, the observed size typically ranges from 110-140 kDa due to post-translational modifications, particularly glycosylation .

• Cross-reactivity testing: Verify reactivity across target species. Different ITGB1 antibodies show varying species reactivity - some react only with human samples, while others cross-react with mouse, rat, pig, or dog samples .

• Multi-application validation: Test the antibody in multiple applications (WB, IHC, IF, FC) to ensure consistent results across different experimental contexts.

How should I design experiments to distinguish between active and total ITGB1?

ITGB1 exists in different conformational states, and experimental design should account for this:

• Select appropriate antibodies: Use conformation-specific antibodies that recognize active ITGB1 (such as clone 9EG7) alongside antibodies that detect total ITGB1 (such as HMβ1–1) .

• Flow cytometry approach: For quantitative assessment, isolate target cells (e.g., keratinocytes from epidermal layer), gate out non-target populations, and analyze cells expressing specific markers (e.g., Itga6) for binding of both active ITGB1 and total ITGB1 antibodies .

• Comparative analysis: Compare the ratio of active to total ITGB1 across different experimental conditions. In the Sharpin-deficient mouse model, quantification showed significantly increased active ITGB1 on the surface of keratinocytes compared to controls, despite similar total ITGB1 levels .

• Tissue context consideration: In tissue sections, evaluate both dermal and epidermal ITGB1 activity separately, as high dermal ITGB1 activity may complicate interpretation of epidermal changes .

What controls are essential when using function-blocking ITGB1 antibodies?

Function-blocking experiments require rigorous controls:

• Isotype control antibody: Always include an isotype-matched control antibody at the same concentration to distinguish specific from non-specific effects. For in vivo studies, administer isotype control antibodies via the same route and schedule .

• Validation of efficacy: Confirm the function-blocking ability of the antibody through cell proliferation assays. For example, in primary mouse keratinocyte cultures, ITGB1 function-blocking antibody significantly reduced cell proliferation compared to isotype control antibody treatment .

• Multiple readouts: Assess multiple biological outcomes. In Sharpin cpdm/cpdm mice, ITGB1 function-blocking antibody reduced epidermal hyperproliferation and thickness but did not affect leukocyte infiltration or inflammation markers .

• Dose-response relationship: Test multiple antibody concentrations to establish the optimal inhibitory concentration with minimal off-target effects.

• Experimental timeframe: Consider the duration of treatment necessary for observing phenotypic changes, particularly in in vivo models where tissue remodeling may require extended treatment periods.

How can I optimize Western blot conditions for ITGB1 detection?

Optimizing Western blot detection of ITGB1 requires attention to several factors:

• Sample preparation: ITGB1 is a transmembrane protein, so effective membrane protein extraction methods are essential. Use appropriate lysis buffers containing detergents suitable for membrane proteins.

• Gel percentage selection: Due to ITGB1's high molecular weight (110-140 kDa observed), use lower percentage gels (7.5%) for better resolution and separation .

• Antibody dilution optimization: Test a range of dilutions to determine optimal signal-to-noise ratio. Published protocols use dilutions ranging from 1:500 to 1:10,000 depending on the specific antibody and sample type .

• Exposure time adjustment: Different exposure times may be needed for optimal visualization. For example, in one study, lanes 1-6 required 81 seconds exposure while lanes 7-8 required only 37 seconds .

• Positive control selection: Include well-characterized cell lines known to express ITGB1, such as HeLa, A431, 293T, 3T3-L1, PC-12, or NIH/3T3 cells .

Why might Western blots show multiple bands or unexpected molecular weights with ITGB1 antibodies?

Multiple factors can contribute to band pattern complexity:

• Post-translational modifications: ITGB1 undergoes extensive glycosylation, resulting in observed molecular weights (110-140 kDa) significantly higher than the predicted size (88 kDa) .

• Proteolytic processing: ITGB1 can undergo proteolytic cleavage during sample preparation or as part of normal cellular processing.

• Alternative splicing: Multiple ITGB1 isoforms exist due to alternative splicing, potentially resulting in additional bands.

• Cell type variations: Different cell types may exhibit varying glycosylation patterns or isoform expression. Western blots have demonstrated slight variations in ITGB1 band patterns across HeLa, A431, 293T, 3T3-L1, PC-12, and NIH/3T3 cell lines .

• Antibody specificity: Some antibodies may recognize both mature and precursor forms of ITGB1, or may cross-react with related integrin family members.

What approach should I take when ITGB1 antibody shows weak or no signal in immunohistochemistry?

When facing detection challenges in immunohistochemistry:

• Optimize antigen retrieval: ITGB1 epitopes may be masked during fixation. Test multiple antigen retrieval methods (heat-induced vs. enzymatic) and buffers (citrate vs. EDTA) to expose epitopes effectively.

• Adjust antibody concentration: If signal is weak, try a higher antibody concentration. Recommended dilutions range from 1:50 to 1:200 for standard IHC protocols, but some antibodies may require concentrations as high as 1:2000 .

• Extend incubation time: Consider overnight primary antibody incubation at 4°C instead of shorter incubations at room temperature.

• Verify tissue reactivity: Ensure the antibody is validated for your specific tissue type. As shown in the search results, antibodies may perform differently in different tissues (e.g., hepatocellular carcinoma vs. colon tissue) .

• Use amplification systems: For weak signals, implement signal amplification methods such as biotin-streptavidin systems or polymer-based detection.

• Consider detection system compatibility: Use detection systems optimized for the primary antibody host species (e.g., LeicaDS9800 BondTM Polymer Refine Detection system was effective with rabbit ITGB1 antibodies) .

How can I address non-specific binding in flow cytometry applications?

To improve specificity in flow cytometry:

• Titrate antibody concentration: The optimal concentration balances specific signal intensity with minimal background staining. Start with manufacturer recommendations and adjust as needed.

• Implement rigorous blocking: Block Fc receptors to prevent non-specific binding, especially when analyzing immune cells.

• Use fluorochrome-matched isotype controls: Include properly matched isotype controls to establish background fluorescence levels.

• Optimize cell preparation: Gentle dissociation methods preserve surface ITGB1 integrity while creating single-cell suspensions necessary for flow cytometry.

• Purification quality matters: Use antibodies purified by size-exclusion chromatography to remove unconjugated antibody and free fluorochrome, reducing background signal .

• Consider compensation: When using multiple fluorochromes, proper compensation is essential, especially with FITC-conjugated ITGB1 antibodies that may have spectral overlap with other common fluorophores .

How can function-blocking ITGB1 antibodies elucidate disease mechanisms?

Function-blocking antibodies provide powerful tools for mechanistic studies:

• Phenotype reversal studies: In Sharpin-deficient mice, which develop skin hyperproliferation, treatment with ITGB1 function-blocking antibodies significantly reduced epidermal hyperproliferation and epidermal thickness, identifying ITGB1 activation as a key disease driver .

• Pathway dissection: Function-blocking antibodies allow researchers to distinguish between inflammation-dependent and adhesion-dependent disease mechanisms. In the Sharpin cpdm/cpdm mouse model, ITGB1 inhibition reduced keratinocyte proliferation without affecting inflammation or immune cell infiltration, demonstrating that these processes can be mechanistically separated .

• Cell-autonomous effects: In vitro treatment of isolated primary keratinocytes with ITGB1 function-blocking antibodies significantly reduced cell proliferation, confirming that effects observed in vivo represent cell-autonomous responses rather than secondary effects .

• Temporal control: Unlike genetic knockouts, antibody-mediated inhibition provides temporal control over ITGB1 function, allowing intervention at specific disease stages.

• Therapeutic potential assessment: Successful disease modification with function-blocking antibodies provides proof-of-concept for developing therapeutic strategies targeting ITGB1.

What considerations are important when selecting ITGB1 antibodies for cross-species studies?

Cross-species experiments require careful antibody selection:

When planning cross-species studies:

• Epitope conservation: Verify that the antibody's target epitope is conserved across your species of interest. For example, some antibodies specifically target amino acids 25-100 or 297-380 of ITGB1, and sequence conservation in these regions determines cross-reactivity .

• Application-specific validation: An antibody may work for one application (e.g., WB) in multiple species but fail in another application (e.g., IHC) for certain species.

• Function-blocking considerations: For function-blocking studies, confirm that the targeted functional epitope has conserved biological significance across species.

• Clone selection: Different monoclonal antibodies (e.g., MEM-101A, 3B6, RM1259) have distinct species reactivity profiles .

• Systematic validation: Always perform species-specific validation before proceeding with comparative studies, regardless of manufacturer claims.

How can ITGB1 antibodies be used to study the relationship between integrin activation and disease progression?

ITGB1 antibodies enable sophisticated studies of integrin activation dynamics:

• Conformation-specific detection: Antibodies recognizing active ITGB1 conformations (like clone 9EG7) allow researchers to track integrin activation status during disease progression. In Sharpin-deficient mice, increased active ITGB1 levels were observed in the epidermis, correlating with hyperproliferation .

• Activation manipulation: Function-blocking antibodies can directly manipulate ITGB1 activation status, allowing researchers to test causality between activation and disease phenotypes.

• Combinatorial genetic models: ITGB1 antibodies can be used in combination with genetic models to dissect complex pathway interactions. For example, studying ITGB1 activation in Tnfr1-Sharpin double knockout mice revealed that ITGB1 hyperactivation occurs independently of TNF receptor signaling .

• Correlative analysis: By combining ITGB1 activation assessment with proliferation markers, researchers identified a direct link between ITGB1 hyperactivation and keratinocyte hyperproliferation .

• Therapeutic intervention studies: Sequential treatment studies using function-blocking antibodies at different disease stages can identify optimal therapeutic windows for intervention.

How might single-cell technologies enhance ITGB1 antibody applications?

Emerging single-cell approaches offer new possibilities:

• Single-cell activation profiling: Using conformation-specific antibodies in single-cell flow cytometry allows researchers to quantify the heterogeneity of ITGB1 activation within populations and identify distinctly responsive subpopulations.

• Multi-parameter analysis: Combining ITGB1 activation markers with lineage-specific markers (like Itga6 for keratinocytes) enables identification of cell type-specific activation patterns within complex tissues .

• FACS-based isolation: Active ITGB1-high versus active ITGB1-low cell populations can be isolated via flow sorting for downstream molecular characterization.

• Spatial analysis integration: Combining spatially-resolved antibody detection with single-cell transcriptomics could reveal microenvironment-dependent ITGB1 activation patterns.

• Longitudinal activation dynamics: With live-cell compatible antibodies, researchers might track ITGB1 activation dynamics in individual cells over time.

What methodological advances might improve ITGB1 antibody specificity and utility?

Several technological approaches could enhance ITGB1 antibody research:

• Recombinant antibody engineering: DNA recombinant technology and in vitro genetic manipulation (as used for the Cusabio ITGB1 recombinant monoclonal antibody) allows creation of highly specific antibodies with reduced batch-to-batch variability .

• Epitope-specific antibody development: Antibodies targeting specific functional domains of ITGB1 could provide more precise mechanistic insights than general ITGB1 antibodies.

• Bifunctional antibodies: Engineering bifunctional antibodies that simultaneously bind ITGB1 and its alpha subunit partners could enable targeting of specific integrin heterodimers.

• Proximity labeling applications: Combining ITGB1 antibodies with proximity labeling technologies could identify context-specific ITGB1 interaction partners.

• Super-resolution compatibility: Developing ITGB1 antibodies compatible with super-resolution microscopy techniques would enable nanoscale visualization of integrin clustering and focal adhesion formation.

How can ITGB1 antibodies contribute to therapeutic development?

ITGB1 antibodies have significant translational potential:

• Target validation: Function-blocking studies provide critical proof-of-concept data. Research using ITGB1 function-blocking antibodies in the Sharpin-deficient mouse model demonstrated that ITGB1 inhibition effectively reduced pathological epidermal hyperproliferation .

• Biomarker development: ITGB1 activation-specific antibodies could serve as prognostic or predictive biomarkers in diseases characterized by aberrant integrin signaling.

• Patient stratification tools: Antibody-based assessment of ITGB1 activation status could identify patient subgroups most likely to benefit from integrin-targeting therapies.

• Therapeutic antibody optimization: Function-blocking antibodies can be humanized and optimized for therapeutic applications, improving affinity, specificity, and pharmacokinetic properties.

• Combination therapy assessment: ITGB1 antibodies can help identify synergistic combinations of integrin inhibition with other targeted therapies or standard treatments.