ITGA5 Antibody

What are the optimal storage conditions for maintaining ITGA5 antibody efficacy?

ITGA5 antibodies should typically be stored at -20°C for long-term preservation. For antibodies in liquid form, storage at 4°C for up to one month is appropriate for frequent use. Most manufacturers recommend avoiding repeated freeze-thaw cycles to maintain antibody performance. For lyophilized antibodies, reconstitution should follow manufacturer guidelines, after which aliquoting and freezing at -20°C is recommended for up to six months . When storing at 4°C for short-term use, antibodies containing preservatives such as sodium azide (typically at 0.02% concentration) help prevent microbial contamination .

What is the expected molecular weight range when detecting ITGA5 using Western blot?

ITGA5 has a calculated molecular weight of approximately 114-115 kDa, but the observed molecular weight typically ranges between 135-150 kDa in Western blot applications . This discrepancy occurs due to post-translational modifications, particularly glycosylation. Some researchers also report detecting smaller fragments (around 19 kDa) that represent cleaved products of ITGA5 . For accurate identification, positive controls from tissues with known high ITGA5 expression (such as placenta) are recommended. Western blot images from multiple sources consistently show the major band at approximately 150 kDa with varying intensity depending on the cell line or tissue type examined .

Which tissue samples are recommended as positive controls for ITGA5 antibody validation?

The most reliable positive controls for ITGA5 antibody validation include:

• Human placenta tissue (consistently high expression across multiple studies)

• Human bladder tissue

• HT-1080 (human fibrosarcoma cell line)

• A549 (human lung carcinoma cell line)

• U937 (human histiocytic lymphoma cell line)

• 3T3-L1 (mouse fibroblast cell line)

These samples consistently demonstrate strong ITGA5 expression and are recommended for initial antibody validation experiments before proceeding to experimental samples .

What dilution ranges are recommended for different applications of ITGA5 antibodies?

The optimal dilution varies by application and antibody source. Based on multiple manufacturer recommendations:

These ranges provide starting points for optimization, but the actual working concentration should be determined empirically for each experimental system . Monoclonal antibodies typically require higher concentrations than polyclonal antibodies for equivalent signal intensity.

How should antigen retrieval be optimized for ITGA5 detection in paraffin-embedded tissue sections?

For optimal ITGA5 detection in formalin-fixed paraffin-embedded (FFPE) tissues, heat-mediated antigen retrieval is crucial. Two primary buffer systems have proven effective:

• Tris-EDTA buffer (pH 9.0): This alkaline pH buffer is the preferred method according to multiple manufacturers, providing superior epitope exposure for many ITGA5 antibodies .

• Citrate buffer (pH 6.0): Some antibodies perform adequately with citrate buffer, but generally with lower signal intensity compared to Tris-EDTA .

The protocol should include:

• Heating to 95-100°C for 15-20 minutes

• Slow cooling to room temperature

• Complete buffer washing before blocking steps

Insufficient antigen retrieval is the most common cause of false negative results in ITGA5 immunohistochemistry, particularly in tissues with dense extracellular matrix .

What blocking conditions minimize background staining when using ITGA5 antibodies?

Effective blocking for ITGA5 antibody applications requires careful consideration of the following parameters:

• Blocking agent: 5-10% normal serum from the species in which the secondary antibody was raised (typically goat) provides optimal blocking. BSA (1-5%) can be used as an alternative .

• Additional blocking components:

  • 0.1-0.3M glycine helps reduce aldehyde-induced background

  • 0.1-0.3% Triton X-100 improves antibody penetration in immunofluorescence applications

  • 0.1% Tween-20 in PBS-based blocking buffers reduces non-specific binding

• Blocking duration: 1 hour at room temperature is standard, though longer incubation (up to 2 hours) may be necessary for tissues with high endogenous biotin or with high natural binding to IgG .

The high specificity of monoclonal anti-ITGA5 antibodies like EPR7854 reduces non-specific binding compared to polyclonal alternatives .

How can researchers verify ITGA5 antibody specificity?

Comprehensive ITGA5 antibody validation should include multiple approaches:

• Genetic validation: Testing antibodies in ITGA5 knockout/knockdown systems provides the strongest evidence for specificity. Several studies demonstrate complete loss of signal in ITGA5 knockout HAP1 cells, confirming antibody specificity .

• Immunoreactivity profile: Testing across multiple species and tissues with known differential expression patterns. Expected cross-reactivity with human, mouse, and rat ITGA5 should be confirmed .

• Molecular weight verification: Confirming the detection of bands at the expected molecular weight range (135-150 kDa) on Western blots .

• Peptide competition assays: Pre-incubation with the immunizing peptide should abolish specific signal. Some manufacturers offer blocking peptides specifically for this purpose .

• Comparing multiple antibody clones: Using antibodies raised against different ITGA5 epitopes should yield consistent staining patterns in the same samples .

Can ITGA5 antibodies distinguish between activated and inactive integrin conformations?

• SNAKA51 clone (not in the search results but known in the field) specifically recognizes the extended, active conformation of α5β1 integrin.

• Function-blocking antibodies like P8D4 recognize composite epitopes on the α5β1 heterodimer and can block binding of α5β1 integrin to the synergy site in the type III-9 fibronectin repeat .

For researchers interested in distinguishing activation states:

• Combine total ITGA5 antibodies with activation-specific markers

• Use functional assays (adhesion, migration) alongside antibody detection

• Consider flow cytometry with multiple antibody clones recognizing different epitopes

It's important to note that conformational specificity must be experimentally verified as few commercial antibodies clearly indicate conformational specificity in their documentation .

How can ITGA5 antibodies be effectively used in studying cancer progression and therapeutic targeting?

ITGA5 is increasingly recognized as an oncogenic biomarker and potential therapeutic target in various cancers, requiring specialized antibody applications:

Research indicates ITGA5 expression correlates with immune checkpoint molecules, suggesting potential combination approaches with immunotherapy .

What are the key considerations when using ITGA5 antibodies for co-immunoprecipitation of integrin complexes?

Co-immunoprecipitation (Co-IP) of ITGA5-containing complexes requires specific methodological considerations:

• Native complex preservation:

  • Use mild detergents (0.5-1% NP-40 or CHAPS) that maintain heterodimer integrity

  • Include divalent cations (1-2 mM Mn²⁺ or Mg²⁺) in lysis buffers to stabilize integrin conformation

  • Avoid reducing agents that disrupt disulfide bonds critical for integrin structure

• Antibody selection:

  • Choose antibodies validated specifically for immunoprecipitation applications

  • Consider antibodies recognizing extracellular domains which typically preserve functional complexes better than those targeting cytoplasmic domains

  • For heterodimer-specific studies, antibodies recognizing composite epitopes (like P8D4) may be advantageous

• Technical optimization:

  • Pre-clear lysates thoroughly to reduce non-specific binding

  • Crosslinking antibodies to beads prevents heavy chain interference in subsequent immunoblotting

  • Extended incubation times (overnight at 4°C) improve complex recovery efficiency

• Controls:

  • Include isotype controls matched to the primary antibody

  • Perform reciprocal IPs (using antibodies against expected binding partners)

  • Include ITGA5-negative cell lines as negative controls

How should researchers interpret discrepancies in ITGA5 detection between different antibody clones?

When confronting discrepancies between different anti-ITGA5 antibody results, several factors should be systematically evaluated:

• Epitope differences:

  • Different antibodies recognize distinct epitopes that may be differentially masked in certain contexts

  • Some epitopes might be inaccessible in particular protein conformations or complexes

  • Post-translational modifications may affect epitope accessibility

• Clonality considerations:

  • Monoclonal antibodies (like EPR7854) offer higher specificity but might miss certain isoforms

  • Polyclonal antibodies provide broader detection but with potential for cross-reactivity

• Methodological variables:

  • Sample preparation methods significantly impact epitope exposure

  • Fixation protocols affect antibody penetration and antigen recognition

  • Application-specific optimizations may be required for the same antibody across different techniques

• Resolution approach:

  • Perform antibody validation using knockout/knockdown controls for each application

  • Compare results with orthogonal detection methods (qPCR, mass spectrometry)

  • Consider using antibody cocktails targeting multiple epitopes for comprehensive detection

What special considerations apply when using ITGA5 antibodies in flow cytometry for cell sorting?

Flow cytometry applications with ITGA5 antibodies require specific optimization strategies:

• Sample preparation:

  • Use enzymatic dissociation methods that preserve surface epitopes (avoid trypsin which can cleave integrins)

  • For adherent cells, EDTA-based dissociation preserves intact integrin complexes

  • Maintain cells at 4°C throughout processing to prevent internalization

• Antibody selection and titration:

  • Choose antibodies specifically validated for flow cytometry applications

  • Optimal concentration range is typically 1:50-1:100 but requires titration for each system

  • Consider directly conjugated antibodies to avoid secondary antibody complications

• Gating strategy optimization:

  • Include viability dyes to exclude dead cells which show non-specific binding

  • Implement appropriate compensation when using multiple fluorophores

  • Consider the heterogeneous expression of ITGA5 when setting sorting gates

• Functional validation:

  • Sorted ITGA5-high and ITGA5-low populations should demonstrate expected functional differences

  • Post-sort analysis of a small aliquot confirms population purity

  • Functional assays (adhesion to fibronectin) validate the biological significance of sorted populations

How can ITGA5 antibodies be utilized in studying cancer stem cell populations?

ITGA5 has emerged as a potential marker for cancer stem-like cells in several tumor types, requiring specialized antibody applications:

• Identification of stem-like populations:

  • Flow cytometry using anti-ITGA5 antibodies in combination with established stem cell markers (CD44, CD133, ALDH) identifies unique subpopulations with enhanced tumorigenic potential

  • Multiparameter analysis should include proper compensation and FMO (fluorescence minus one) controls

• Functional characterization:

  • Immunomagnetic separation using ITGA5 antibodies allows isolation of live cells for functional assays

  • Sorted ITGA5-high versus ITGA5-low populations can be compared for sphere formation, self-renewal, and tumor initiation capacity

  • Blocking antibodies can assess the functional requirement of ITGA5 in maintaining stemness properties

• Therapeutic targeting:

  • Function-blocking ITGA5 antibodies potentially disrupt cancer stem cell niches

  • Combining ITGA5 inhibition with conventional therapies targets both bulk tumor and stem-like populations

  • Treatment efficacy can be monitored through quantitative changes in ITGA5-positive stem-like fractions

This approach is particularly relevant in gliomas and pancreatic cancer, where ITGA5 expression correlates with aggressive disease features and therapeutic resistance .

What considerations apply when using ITGA5 antibodies to study the tumor microenvironment?

ITGA5 plays crucial roles in tumor-stroma interactions, requiring specific approaches for microenvironment analysis:

• Cell type-specific detection:

  • Dual immunofluorescence combining ITGA5 with cell type-specific markers (α-SMA for activated fibroblasts, CD31 for endothelial cells, CD45 for immune cells) distinguishes ITGA5 expression in different compartments

  • Spectral unmixing may be necessary to resolve overlapping signals in multiplexed approaches

• Spatial analysis:

  • Whole-slide imaging with ITGA5 antibodies maps expression patterns across different tumor regions

  • Quantitative spatial analysis correlates ITGA5 distribution with invasion fronts, hypoxic regions, and immune-rich areas

  • Digital pathology tools can quantify ITGA5-positive cell densities in distinct microenvironment niches

• Extracellular matrix interactions:

  • Co-staining for ITGA5 and ECM components (fibronectin, collagen) visualizes functional adhesion sites

  • In situ proximity ligation assays detect specific ITGA5-ECM protein interactions with molecular precision

  • Correlative microscopy combining immunofluorescence with electron microscopy provides ultrastructural context

Research demonstrates that ITGA5 inhibition in pancreatic stellate cells attenuates desmoplasia and improves therapeutic outcomes, highlighting the importance of stromal ITGA5 targeting .

How should researchers design experiments to study ITGA5-mediated signaling pathways?

Investigating ITGA5-mediated signaling requires multifaceted approaches:

• Activation-dependent signaling:

  • Use ITGA5 antibodies that either block or preserve integrin activation to distinguish activation-dependent signals

  • Compare signaling responses when cells are plated on specific ITGA5 ligands (fibronectin) versus non-specific substrates

  • Time-course experiments track rapid phosphorylation events downstream of ITGA5 engagement

• Complex formation analysis:

  • Co-immunoprecipitation with ITGA5 antibodies followed by immunoblotting for known signaling partners (FAK, Src, ILK)

  • Proximity ligation assays visualize specific ITGA5-partner interactions with subcellular resolution

  • Mass spectrometry of ITGA5 immunoprecipitates identifies novel interaction partners and post-translational modifications

• Functional intervention:

  • Function-blocking ITGA5 antibodies disrupt specific ligand interactions

  • shRNA-mediated ITGA5 knockdown models show attenuated α-SMA expression and reduced activation of downstream pathways

  • Peptide inhibitors (like the novel AV3 peptidomimetic) provide alternative approaches to antibody-based blocking

Research indicates ITGA5 signaling plays critical roles in TGF-β-induced cellular activation, with knockdown of ITGA5 in pancreatic stellate cells reducing α-SMA and collagen I expression .

What methodological approaches allow measurement of ITGA5 activation states in living cells?

Studying ITGA5 activation dynamics in living cells requires specialized techniques:

• Conformation-sensitive antibodies:

  • Live-cell compatible antibodies that recognize specific activation epitopes

  • Non-function-blocking antibodies preserve normal cellular activities while reporting conformational states

  • Fab fragments minimize integrin crosslinking that could artificially alter activation

• FRET-based biosensors:

  • Integrin tension sensors based on FRET pairs inserted into ITGA5 cytoplasmic domains

  • Conformational biosensors that report on extension or clustering states

  • Live imaging captures real-time activation dynamics during cell migration or matrix remodeling

• Ligand-binding assays:

  • Fluorescently labeled soluble fibronectin fragments quantify active ITGA5 levels

  • Flow cytometry with activation-specific antibodies measures population-level changes

  • Quantitative binding kinetics distinguish affinity modulation from receptor number changes

• Complementary approaches:

  • Total internal reflection fluorescence (TIRF) microscopy visualizes ITGA5 clustering at adhesion sites

  • Fluorescence recovery after photobleaching (FRAP) measures mobility changes associated with activation

  • Super-resolution microscopy resolves nanoscale organization of active versus inactive integrins