ITGB6 Antibody, Biotin conjugated

What is ITGB6 and what cellular processes does it mediate?

ITGB6 (integrin subunit beta 6) is a 788-amino acid protein that belongs to the Integrin beta chain family. It is primarily membrane-associated and features several glycosylation sites. ITGB6 typically pairs with integrin alpha V (ITGAV) to form the αVβ6 heterodimer, which functions in cell adhesion, signaling, and tissue remodeling pathways . The integrin αVβ6 heterodimer has a calculated molecular weight of approximately 114.7 kDa (ITGAV) and 79.6 kDa (ITGB6), though gel migration patterns may show bands at 135-150 kDa and 82-95 kDa respectively under non-reducing conditions . As a transmembrane receptor, ITGB6 plays crucial roles in epithelial cell-extracellular matrix interactions, wound healing, and TGF-β activation, making it particularly relevant in fibrosis and cancer research.

What distinct advantages do biotin-conjugated ITGB6 antibodies offer over unconjugated versions?

Biotin-conjugated ITGB6 antibodies provide several methodological advantages for research applications. The biotin-avidin/streptavidin interaction has an exceptionally high affinity (Kd ≈ 10^-15 M), enabling sensitive detection systems with amplified signals. This conjugation allows for versatile detection methods as the biotin tag can be recognized by various labeled streptavidin conjugates (fluorescent, enzymatic, or gold-labeled) . Additionally, biotin conjugation maintains antibody functionality while providing flexibility in detection systems without requiring direct labeling of the primary antibody. This is particularly valuable in multilabeling experiments where several biomarkers need to be detected simultaneously, as different biotin-conjugated primary antibodies can be used with distinguishable streptavidin-conjugated detection systems.

What are the validated applications for biotin-conjugated ITGB6 antibodies?

Based on the available research data, biotin-conjugated ITGB6 antibodies have been validated for multiple research applications:

Several manufacturers specifically highlight western blot and immunocytochemistry as primary applications for biotin-conjugated mouse ITGB6 antibodies . These applications leverage the signal amplification capabilities of the biotin-streptavidin system, particularly beneficial when detecting low-abundance integrin expression.

What species reactivity profiles are available for ITGB6 antibodies?

ITGB6 antibodies display varied species reactivity profiles depending on the specific clone and manufacturer:

According to the search results, several manufacturers offer ITGB6 antibodies with human, mouse, and rat reactivity . For biotin-conjugated versions specifically, mouse-reactive and human-reactive options are most commonly available. When selecting an antibody for cross-species studies, researchers should verify epitope conservation and cross-reactivity validation data rather than relying solely on manufacturer claims.

What are the optimal sample preparation methods for biotin-conjugated ITGB6 antibody applications?

Sample preparation requirements vary by application, but several methodological considerations are critical for biotin-conjugated ITGB6 antibody use:

For Western Blot:

• Use fresh or properly stored (-80°C) samples with protease inhibitors

• Perform membrane protein enrichment for enhanced sensitivity given ITGB6's membrane localization

• Standard RIPA or NP-40 lysis buffers with proper detergent concentrations preserve integrin structure

• Non-reducing conditions may better preserve the native conformation of ITGB6

• When using reducing conditions, migration patterns of 79.6 kDa for ITGB6 should be expected, though glycosylation may result in higher apparent molecular weights

For Immunohistochemistry:

• Both frozen and formalin-fixed paraffin-embedded (FFPE) tissues can be used

• FFPE tissues typically require antigen retrieval (heat-induced epitope retrieval in citrate buffer pH 6.0)

• Include enzymatic biotin blocking steps to reduce endogenous biotin background

• Optimal fixation: 10% neutral buffered formalin for 24-48 hours for consistent epitope preservation

How can researchers optimize detection systems for biotin-conjugated ITGB6 antibodies?

Optimization strategies should consider both signal enhancement and background reduction:

Signal Enhancement:

• Use streptavidin-HRP or streptavidin-AP systems for chromogenic detection with amplification

• For fluorescence detection, streptavidin conjugated to bright, photostable fluorophores (Alexa Fluor 488, 555, 647) provides optimal results

• Tyramide signal amplification (TSA) can be combined with biotin-streptavidin systems for ultrasensitive detection

• For low-expression studies, consider multistep amplification using biotin-streptavidin-biotin laddering techniques

Background Reduction:

• Implement rigorous biotin blocking protocols using avidin-biotin blocking kits

• For tissue samples, include blocking of endogenous biotin using avidin followed by biotin

• Use casein-based blockers rather than BSA-based blockers when possible (less biotin contamination)

• Titrate antibody concentrations carefully, as excess biotin-conjugated antibody increases non-specific binding

What controls should be included in experiments utilizing biotin-conjugated ITGB6 antibodies?

A robust experimental design requires multiple controls to ensure reliability:

Essential Controls:

• Isotype control with matching biotin conjugation to assess non-specific binding

• Blocking peptide/antigen competition assay to verify antibody specificity

• ITGB6 knockout or knockdown samples as negative controls

• Known ITGB6-positive samples (e.g., certain epithelial cell lines) as positive controls

• Secondary-only (streptavidin-only) controls to assess background from the detection system

• Endogenous biotin blocking controls to evaluate blocking efficiency

Advanced Validation Controls:

• Orthogonal detection method comparison (e.g., comparing protein levels with mRNA expression)

• Independent antibody validation using two antibodies recognizing different epitopes

• Phosphatase treatment for phospho-specific applications to confirm specificity

How should dilution optimization be performed for biotin-conjugated ITGB6 antibodies?

Methodical dilution optimization is crucial for balancing signal strength and specificity:

• Begin with the manufacturer's recommended dilution range (typically 1:100 to 1:500 for most applications)

• Perform a systematic dilution series covering at least 3-4 dilutions above and below the recommended range

• Use consistent positive control samples with known ITGB6 expression levels

• Evaluate both signal intensity and background levels at each dilution

• Calculate signal-to-noise ratios to identify optimal working dilution

• Verify reproducibility by repeating optimal dilution in independent experiments

• For quantitative applications, ensure the selected dilution falls within the linear dynamic range of detection

How can biotin-conjugated ITGB6 antibodies be effectively used in multiplex immunofluorescence?

Multiplex strategies require careful planning to leverage biotin conjugation advantages:

Methodological Approach:

• Design a sequential staining strategy where the biotin-conjugated ITGB6 antibody is applied in a separate round from other biotin-conjugated antibodies

• Employ thorough biotin blocking between rounds using concentrated biotin solution (1-2 mg/ml)

• Use spectrally distinct fluorophore-conjugated streptavidin for each biotin-antibody round

• Consider tyramide signal amplification (TSA) with subsequent antibody stripping for highest multiplexing capacity

• When using with αV integrin staining, select antibodies recognizing the heterodimer (αVβ6) specifically rather than individual subunits to prevent misleading co-localization artifacts

Recommended Panel Design:

• Round 1: Biotin-ITGB6 + Streptavidin-Fluorophore 1

• Complete biotin blocking

• Round 2: Direct fluorophore-conjugated antibodies for additional markers

• Nuclear counterstain in final round

What are the key considerations for using biotin-conjugated ITGB6 antibodies in flow cytometry?

Flow cytometry applications require specific methodological adjustments:

Protocol Optimizations:

• Use gentle cell dissociation methods (EDTA-based, enzyme-free when possible) to preserve membrane integrity and integrin epitopes

• Maintain cells at 4°C throughout staining to prevent integrin internalization

• Include cations (Ca²⁺, Mg²⁺) in staining buffers to maintain integrin conformational integrity

• Apply sequential staining: primary biotin-ITGB6 antibody followed by fluorochrome-conjugated streptavidin

• Include viability dye to exclude dead cells which can bind antibodies non-specifically

• For intracellular staining, use gentle permeabilization (0.1% saponin) to preserve membrane structures

Gating Strategy:

• Forward/side scatter to identify intact cells

• Viability dye exclusion gate

• Single cell gate using FSC-H vs FSC-A

• ITGB6-positive population analysis with fluorescence minus one (FMO) controls

How should researchers troubleshoot non-specific binding issues with biotin-conjugated ITGB6 antibodies?

Systematic troubleshooting approaches can resolve common technical challenges:

When encountering persistent non-specific binding, implementing a dual-detection strategy can help distinguish true signal from artifact. For example, using a directly-labeled antibody against another epitope of ITGB6 or its binding partner ITGAV can confirm the specificity of signals observed with the biotin-conjugated antibody .

What advanced quantification methods are appropriate for biotin-conjugated ITGB6 antibody data?

Quantitative analysis requires rigorous methodological approaches:

For Western Blot:

• Densitometric analysis with normalization to loading controls

• Standard curves using recombinant protein for absolute quantification

• Consider the non-linear nature of chemiluminescent detection when quantifying

• Validate quantification across multiple exposure times to ensure measurements within linear range

For Flow Cytometry:

• Mean/median fluorescence intensity (MFI) measurements with background subtraction

• Molecules of Equivalent Soluble Fluorochrome (MESF) calibration for standardized quantification

• Population frequency analysis with clearly defined positive thresholds based on FMO controls

For Immunohistochemistry/Immunofluorescence:

• Digital image analysis with automated thresholding

• H-score calculation (percentage of positive cells × intensity scoring)

• Machine learning approaches for pattern recognition and intensity quantification

• Spatial analysis of membrane localization patterns

How are biotin-conjugated ITGB6 antibodies being utilized in cancer research?

ITGB6 has emerged as an important biomarker and therapeutic target in multiple cancer types:

Key Research Applications:

• Diagnostic biomarker evaluation in carcinomas (particularly oral, pancreatic, colon, and breast)

• Correlation of ITGB6 expression with invasive phenotypes and patient outcomes

• Monitoring epithelial-to-mesenchymal transition (EMT) processes

• Development of targeted therapies against ITGB6-expressing tumors

• Imaging probe development for ITGB6-positive tumor detection

Biotin-conjugated ITGB6 antibodies provide methodological advantages in these applications through signal amplification capabilities and compatibility with multiple detection systems. They're particularly valuable in tissue microarray studies where sensitivity and specificity are paramount for analyzing large cohorts of patient samples with potentially variable ITGB6 expression levels.

What is the significance of ITGB6 in fibrosis research and how can biotin-conjugated antibodies contribute to this field?

ITGB6 plays a critical role in fibrotic processes through TGF-β activation:

Methodological Approaches:

• Dual immunofluorescence to correlate ITGB6 expression with activated TGF-β signaling

• Temporal expression analysis during fibrosis progression using biotin-conjugated antibodies

• Co-localization studies with ECM components to map ITGB6 distribution in fibrotic tissues

• In vitro blocking studies to assess therapeutic targeting potential

The signal amplification provided by biotin-conjugated antibodies is particularly valuable when studying early fibrotic changes where ITGB6 upregulation may be subtle. Additionally, the compatibility with multiple detection systems allows for temporal studies where different markers can be visualized in the same tissue sections at different timepoints.

How can biotin-conjugated ITGB6 antibodies be used to study integrin heterodimer formation and function?

ITGB6 primarily functions as a heterodimer with integrin αV (ITGAV):

Experimental Approaches:

• Co-immunoprecipitation studies to isolate intact heterodimers

• Proximity ligation assays (PLA) to visualize heterodimer formation in situ

• FRET analysis to study molecular interactions between αV and β6 subunits

• Competitive binding studies to analyze ligand specificity

Biotin-conjugated ITGB6 antibodies can be particularly useful in pull-down assays where the high-affinity biotin-streptavidin interaction provides efficient isolation of integrin complexes. Specialized antibodies recognizing the intact heterodimer (αVβ6) rather than individual subunits offer additional research opportunities for studying the functional complex as a unified entity .

What emerging technologies are enhancing the utility of biotin-conjugated ITGB6 antibodies?

Several technological advancements are expanding research capabilities:

Advanced Imaging Technologies:

• Super-resolution microscopy (STORM, PALM) for nanoscale localization studies

• Expansion microscopy for enhanced spatial resolution of integrin distribution

• Light sheet microscopy for 3D imaging of integrin patterns in tissue samples

• Mass cytometry (CyTOF) for high-dimensional analysis of integrin expression in complex cell populations

Molecular Engineering Approaches:

• Biotin-conjugated antibody fragments (Fab, scFv) for improved tissue penetration

• Site-specific biotinylation for consistent orientation and performance

• Bispecific antibodies targeting both ITGB6 and functional partners

• Bioorthogonal click chemistry approaches for in situ labeling applications

These emerging technologies enable researchers to ask increasingly sophisticated questions about ITGB6 biology, particularly regarding its spatial organization, molecular interactions, and functional dynamics in complex biological systems.

How should researchers select the most appropriate biotin-conjugated ITGB6 antibody for their specific application?

Selection should follow a systematic evaluation process:

Critical Selection Criteria:

• Validated Applications: Verify the antibody has been specifically validated for your intended application (WB, IHC, FC, etc.)

• Species Reactivity: Confirm reactivity with your experimental model organism

• Epitope Location: Consider functional domains (extracellular vs intracellular epitopes)

• Clone Type: Evaluate monoclonal vs polyclonal options based on your specific needs

• Validation Data: Review available literature citations and validation figures

• Biotin Conjugation Method: Consider site-specific vs random conjugation approaches

• Lot-to-Lot Consistency: Assess quality control documentation from manufacturers

Decision Matrix for Antibody Selection:

What are the comparative advantages of different biotin conjugation strategies for ITGB6 antibodies?

The biotin conjugation methodology significantly impacts antibody performance:

Conjugation Approaches Comparison:

The search results indicate that some ITGB6 antibody products feature sophisticated biotinylation approaches, including Avitag™ technology that enables site-specific biotinylation . This approach provides consistent orientation and accessibility of the biotin molecule, potentially enhancing detection sensitivity and reproducibility.

How do detection systems compare when using biotin-conjugated ITGB6 antibodies?

Different detection systems offer distinct advantages for various applications:

Detection System Comparison:

For applications requiring the highest sensitivity, TSA amplification systems offer significant advantages but may require additional optimization to control background signal. For multiplexed detection, spectrally distinct fluorophore-conjugated streptavidin provides the best balance of sensitivity and multiplexing capability.

How should researchers interpret variable expression patterns of ITGB6 detected by biotin-conjugated antibodies?

Interpretation requires consideration of biological and technical factors:

Biological Variability:

• ITGB6 exhibits context-dependent expression based on cellular differentiation state

• Expression patterns vary between normal and pathological tissues (particularly in fibrosis and cancer)

• Subcellular localization may shift between membrane and cytoplasmic compartments during cellular activation

• Heterogeneous expression within tissues often reflects functional specialization

Technical Considerations:

• Signal intensity variations should be calibrated against validated positive controls

• Membrane staining patterns should predominate for intact cells with proper epitope preservation

• Cytoplasmic signal may represent newly synthesized protein, internalized receptor, or fixation artifact

• Correlation with mRNA expression data provides validation of protein-level findings

When interpreting heterogeneous expression patterns, quantitative analysis addressing both staining intensity and percentage of positive cells provides more comprehensive characterization than binary positive/negative classifications.

What are the recommended approaches for quantitative analysis of ITGB6 expression using biotin-conjugated antibodies?

Quantitative analysis requires appropriate methodologies for each application:

For Tissue Section Analysis:

• Digitize stained sections at consistent exposure settings

• Implement automated detection algorithms with manual verification

• Quantify based on:

  • Percentage of positive cells in defined regions

  • Staining intensity (0, 1+, 2+, 3+)

  • Combined H-score (percentage × intensity)

  • Membrane completeness scores for cell surface molecules

For Western Blot Quantification:

• Use standard curves with recombinant protein when possible

• Normalize to appropriate loading controls

• Ensure measurements are taken within the linear range of detection

• Average results from multiple independent experiments

For Flow Cytometry:

• Report both percentage of positive cells and mean/median fluorescence intensity

• Use calibration beads for standardized measurements

• Include appropriate controls for autofluorescence and non-specific binding

Regardless of the approach, statistical analysis should account for biological variability and include appropriate tests for significance assessment.

How can researchers validate the specificity of biotin-conjugated ITGB6 antibody staining patterns?

Multiple validation approaches should be implemented:

Essential Validation Methods:

• Genetic Validation: Compare staining in ITGB6 knockout/knockdown vs. wild-type samples

• Peptide Competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple Antibody Concordance: Compare staining patterns with independent antibodies targeting different ITGB6 epitopes

• Orthogonal Method Correlation: Compare protein detection with mRNA expression (ISH or qPCR)

• Expected Distribution Analysis: Verify staining in tissues with known ITGB6 expression patterns

Advanced Validation Approaches:

• Correlation with functional readouts (e.g., TGF-β activation)

• Mass spectrometry validation of immunoprecipitated proteins

• Comparison with recombinant expressed protein as positive control

• Cross-species conservation analysis of staining patterns

The search results indicate several manufacturers provide validation figures from publications that can serve as reference points for expected staining patterns across different applications .