ITGB3 (Ab-773) Antibody

What is ITGB3 and why is it significant in research?

ITGB3 (Integrin Subunit Beta 3) is a protein-coding gene that encodes a 788-amino acid residue protein localized to the cell membrane. The protein functions as a subunit of integrin complexes and is involved in critical biological processes including cell adhesion and apoptotic pathways. It features phosphorylated and glycosylated post-translational modifications and is predominantly expressed in bone marrow . ITGB3 has significant research importance due to its association with multiple diseases including Bleeding Disorder, Platelet-Type, 24 and Glanzmann Thrombasthenia 2 . The protein's involvement in Signaling downstream of RAS mutants and Apoptotic Pathways in Synovial Fibroblasts makes it a target of interest in both basic and translational research . Additionally, ITGB3 has been implicated in neurological functions, with evidence of expression in the human brain and roles in synaptogenesis and neurogenesis .

How does the ITGB3 (Ab-773) Antibody differ from other anti-ITGB3 antibodies?

The ITGB3 (Ab-773) Antibody specifically targets the phosphorylated form of ITGB3 at residue 773, distinguishing it from other anti-ITGB3 antibodies that may target different epitopes or modified states of the protein . This antibody exhibits cross-reactivity with human, mouse, and rat specimens, making it versatile for comparative studies across these species . Its applications include Western Blot (WB) and Immunohistochemistry (IHC), providing flexibility for different experimental approaches . Compared to other antibodies targeting ITGB3, the Ab-773 antibody offers specific recognition of a post-translationally modified form that may be particularly relevant for studying activated states of the protein in various physiological and pathological contexts.

What are the known aliases and related terminology for ITGB3?

ITGB3 is known by several aliases including GP3A, which is commonly used in antibody development and cataloging . Other synonyms include BDPLT16, BDPLT2, and BDPLT24, which reflect its association with bleeding disorders and platelet function . The protein is also sometimes referred to as CD61 in the context of cell surface marker identification, particularly in hematological research. In receptor complexes, ITGB3 pairs with either integrin alpha-v to form the vitronectin receptor (αvβ3) or with integrin alpha-IIb to form the fibrinogen receptor (αIIbβ3) . These different nomenclatures reflect the diverse research contexts in which ITGB3 is studied, from platelet biology to cell adhesion, cancer research, and neuroscience.

What are the validated applications for ITGB3 (Ab-773) Antibody in research?

The ITGB3 (Ab-773) Antibody has been validated for Western Blot (WB) and Immunohistochemistry (IHC) applications, making it suitable for both protein expression quantification and localization studies . In Western Blot applications, this antibody can detect the ITGB3 protein at approximately 87-90 kDa, corresponding to the mature processed form of the protein. For IHC applications, the antibody can be used on formalin-fixed, paraffin-embedded tissues as well as frozen sections, providing flexibility in sample preparation approaches . The antibody's cross-reactivity with human, mouse, and rat specimens expands its utility across different model systems . While not explicitly validated, researchers have also explored its potential applications in immunoprecipitation (IP) and flow cytometry, particularly when studying ITGB3 in complex with other integrin subunits.

How should researchers optimize Western Blot protocols when using ITGB3 (Ab-773) Antibody?

For optimal Western Blot results with ITGB3 (Ab-773) Antibody, researchers should consider several critical factors. Sample preparation should include appropriate lysis buffers that preserve protein integrity while efficiently extracting membrane-bound proteins like ITGB3 . A recommended approach involves using RIPA buffer supplemented with protease and phosphatase inhibitors, particularly when studying the phosphorylated form recognized by this antibody. Protein separation should be performed using 8-10% SDS-PAGE gels to provide optimal resolution for the 87-90 kDa ITGB3 protein . For transfer, a semi-dry or wet transfer system with methanol-containing transfer buffer works well for this membrane protein. Blocking should be performed with 5% non-fat dry milk or BSA in TBST, with BSA preferred when detecting phosphorylated forms. The primary antibody dilution range typically falls between 1:500 to 1:2000, with overnight incubation at 4°C producing the best signal-to-noise ratio. Signal detection using either chemiluminescence or fluorescence methods is suitable, with the latter offering better quantification capabilities.

What considerations are important for immunohistochemistry applications of this antibody?

When performing immunohistochemistry with ITGB3 (Ab-773) Antibody, several methodological considerations are critical. Antigen retrieval is essential since formalin fixation can mask epitopes; heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically effective for ITGB3 detection . The antibody works best at dilutions ranging from 1:100 to 1:500, with optimization recommended for each tissue type and fixation method. A detection system based on polymer-HRP is preferable to avidin-biotin methods due to the potential for endogenous biotin interference, particularly in bone marrow samples where ITGB3 is highly expressed . Counterstaining with hematoxylin provides good nuclear contrast without obscuring membrane staining patterns. For fluorescent detection, this antibody can be paired with anti-ITGAV antibodies to visualize the αvβ3 integrin complex, which requires careful selection of secondary antibodies to avoid cross-reactivity. Negative controls should include both isotype controls and peptide competition assays to confirm specificity of the observed staining patterns.

What are common issues when using ITGB3 (Ab-773) Antibody and how can they be resolved?

Researchers working with ITGB3 (Ab-773) Antibody may encounter several technical challenges. One common issue is weak or absent signal in Western blots, which can be addressed by increasing protein loading (50-100 μg recommended), optimizing antibody concentration, extending incubation times, or enhancing detection sensitivity . High background is another frequent problem, typically resolved by more stringent washing steps, reducing antibody concentration, or switching blocking reagents from milk to BSA. Non-specific bands may appear due to protein degradation or cross-reactivity; using fresher samples with added protease inhibitors and performing peptide competition assays can help identify specific signals . For IHC applications, variable staining intensity across different tissue samples can be normalized by standardizing fixation times and antigen retrieval conditions. Membrane proteins like ITGB3 may show extraction difficulties, which can be overcome by using specialized membrane protein extraction buffers containing mild detergents like NP-40 or Triton X-100. For phospho-specific detection, samples should be processed rapidly and maintained with phosphatase inhibitors throughout to preserve the phosphorylated state of ITGB3 at residue 773.

How can researchers validate the specificity of ITGB3 (Ab-773) Antibody results?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For ITGB3 (Ab-773) Antibody, several validation approaches are recommended. Peptide competition assays using the immunizing peptide can confirm binding specificity by demonstrating signal reduction when the antibody is pre-incubated with excess peptide . Genetic validation through siRNA/shRNA knockdown of ITGB3 or CRISPR-Cas9 knockout models provides strong evidence of specificity when the signal disappears in these samples. Comparative analysis with alternative antibodies targeting different ITGB3 epitopes can confirm target recognition patterns. For phospho-specific validation, treatment of samples with phosphatases should eliminate the signal detected by this antibody. Cross-species reactivity testing can verify the conservation of the epitope and validate the antibody's utility in different model organisms . Positive control samples known to express ITGB3, such as platelets or endothelial cells, should be included in all experiments. Mass spectrometry validation of immunoprecipitated proteins can provide definitive evidence of the antibody's target specificity and detection capabilities.

What sample preparation techniques are recommended for different tissue and cell types?

Optimal sample preparation for ITGB3 (Ab-773) Antibody varies by sample type and application. For cell lines, direct lysis in RIPA or NP-40 buffer supplemented with protease and phosphatase inhibitors is effective, with 30-minute extraction on ice followed by centrifugation at 14,000g to remove debris . Tissue samples require more extensive processing: fresh tissues should be minced and homogenized in buffer using a Dounce homogenizer, while frozen tissues benefit from pulverization under liquid nitrogen before adding lysis buffer. For bone marrow samples, where ITGB3 is prominently expressed, red blood cell lysis should be performed before protein extraction . For IHC applications, formalin fixation time should be limited to 24 hours and followed by proper paraffin embedding to preserve antigenicity. For membrane protein enrichment, sucrose gradient ultracentrifugation or commercial membrane protein extraction kits can improve detection sensitivity. Platelets require special handling with prostacyclin to prevent activation during isolation, as activation status significantly affects ITGB3 phosphorylation states. For co-immunoprecipitation studies investigating ITGB3's interactions with other proteins, gentler lysis conditions using buffers with 1% digitonin or 0.5% NP-40 better preserve protein-protein interactions.

How does miRNA regulation of ITGB3 impact antibody-based detection methods?

miRNA regulation of ITGB3 presents important implications for antibody-based detection methods. Research has demonstrated that ITGB3 is directly regulated by several microRNAs, particularly miR-221/222, which bind to the 3'UTR region of ITGB3 mRNA . This post-transcriptional regulation can create discrepancies between mRNA and protein levels, confounding the interpretation of research results. When using ITGB3 (Ab-773) Antibody in cell models with altered miRNA expression, researchers should consider how these regulatory mechanisms affect protein abundance and phosphorylation states . For example, cells grown in serum-free media show dramatically increased ITGB3 expression (3.5-fold) with concurrent downregulation of miR-221 by 2.2-fold, suggesting inverse regulatory relationships . This dynamic regulation may affect epitope accessibility or post-translational modifications detected by the antibody. To account for these effects, researchers should complement antibody-based detection with mRNA quantification and miRNA profiling. Additionally, experimental manipulations of miRNA levels should be considered when validating antibody specificity, as altered miRNA regulation may change the protein's abundance, localization, or modification state in ways that affect antibody recognition.

What is the relationship between ITGB3 and neurological function, and how can the antibody be used to study this?

ITGB3 has emerging roles in neurological function that can be investigated using ITGB3 (Ab-773) Antibody. Studies have shown that ITGB3 is expressed in the human brain and is implicated in synaptogenesis and neurogenesis . The protein plays roles in synaptic plasticity in neuronal hippocampal cultures and regulates excitatory synaptic strength and AMPA receptor expression . ITGB3 (Ab-773) Antibody can be utilized to study these functions through several approaches. Immunohistochemistry of brain sections can map the distribution of ITGB3 across different neural structures and cell types, while co-staining with markers for neurons, glia, and synaptic proteins can reveal specific localization patterns . In primary neuronal cultures, the antibody can be used to track ITGB3 redistribution during synaptic plasticity events using confocal microscopy. For biochemical analyses, the antibody can isolate ITGB3-containing protein complexes from brain tissue through immunoprecipitation, followed by mass spectrometry to identify neurologically relevant interaction partners. Western blot analysis of brain-region-specific samples can quantify ITGB3 expression differences between conditions or disease models . Importantly, since ITGB3 interacts with cell adhesion molecule CHL1 at the cell membrane, double-labeling immunofluorescence with antibodies against both proteins can visualize their co-localization at synaptic sites and reveal how their interaction might be implicated in SSRI antidepressant mechanisms .

How can ITGB3 (Ab-773) Antibody be used to study receptor complex formation?

ITGB3 (Ab-773) Antibody offers valuable approaches for investigating receptor complex formation involving integrin beta-3. ITGB3 forms heterodimeric receptor complexes with either integrin alpha-v (forming αvβ3, the vitronectin receptor) or with integrin alpha-IIb (forming αIIbβ3, the fibrinogen receptor) . Additionally, ITGB3 has been shown to assemble into a heterocomplex with IGF1R, which is essential for IGF-1 signaling . To study these complex formations, researchers can employ several antibody-based techniques. Co-immunoprecipitation using ITGB3 (Ab-773) Antibody can pull down intact receptor complexes from cell lysates, which can then be analyzed for partner proteins by Western blotting . Proximity ligation assays (PLA) combine the antibody with antibodies against potential binding partners to visualize protein interactions with nanometer resolution in situ. Förster resonance energy transfer (FRET) microscopy using fluorescently labeled antibodies can detect molecular proximity between ITGB3 and its binding partners. Blue native PAGE followed by Western blotting with the antibody can separate and identify native protein complexes containing ITGB3. For therapeutic development, the antibody can monitor changes in receptor complex formation following drug treatments, such as SSRI antidepressants which have been linked to ITGB3 function . Finally, cross-linking mass spectrometry approaches can be combined with immunopurification using this antibody to map the interaction interfaces within ITGB3-containing complexes at amino acid resolution.

How should researchers quantify and normalize ITGB3 expression data from Western blots?

Proper quantification and normalization of ITGB3 Western blot data requires rigorous methodological approaches. When using ITGB3 (Ab-773) Antibody for quantitative analyses, researchers should implement a standardized protocol that includes running a dilution series of a reference sample to establish the linear detection range of the antibody . Densitometric analysis should be performed using software that can accurately subtract background signals and define band boundaries consistently across samples. For normalization, loading controls should be selected carefully; for membrane proteins like ITGB3, traditional housekeeping proteins like GAPDH or β-actin may not accurately reflect loading, and membrane-specific controls such as Na+/K+-ATPase or cadherin are preferable . When studying phosphorylated ITGB3, normalization to total ITGB3 levels rather than housekeeping proteins provides more meaningful data about the activation state. This requires running parallel blots or stripping and reprobing with antibodies against total ITGB3. The calculation should follow this formula:

$\text{Relative phospho-ITGB3 expression} = \frac{(\text{Phospho-ITGB3 band intensity})}{(\text{Total ITGB3 band intensity})} \times \text{Normalization factor}$

Statistical analysis should include multiple biological replicates (n≥3) and appropriate tests based on data distribution. For comparing multiple treatment groups, ANOVA with post-hoc tests is recommended over multiple t-tests to control for family-wise error rates.

What criteria should be used to evaluate ITGB3 staining patterns in immunohistochemistry?

Evaluating ITGB3 staining patterns in immunohistochemistry requires systematic assessment based on established criteria. When using ITGB3 (Ab-773) Antibody for IHC, researchers should implement a standardized scoring system that considers both staining intensity and distribution patterns . Staining intensity should be graded on a scale (0=negative, 1=weak, 2=moderate, 3=strong), while distribution can be quantified as percentage of positive cells in defined fields. ITGB3 typically exhibits membrane localization, with potential cytoplasmic staining representing internalized or newly synthesized protein . A composite H-score can be calculated using the formula:

$\text{H-score} = \sum (i \times \text{Pi})$

where i is the intensity score and Pi is the percentage of cells with that intensity (0-100%).

Cell-type specific staining patterns should be noted, as ITGB3 expression varies across cell types within a tissue. In blood vessels, endothelial cells typically show moderate to strong membrane staining, while in bone marrow, megakaryocytes and platelets exhibit intense staining . Stromal components and extracellular matrix may show variable staining that should be distinguished from specific cellular labeling. For phospho-specific detection using ITGB3 (Ab-773) Antibody, activated versus quiescent cells often show differential staining intensities. Digital image analysis using specialized software can provide more objective quantification compared to manual scoring, particularly for co-localization studies with other markers. When comparing pathological versus normal tissues, changes in both intensity and subcellular localization should be documented and correlated with clinical or experimental parameters.

How can researchers integrate ITGB3 data with other molecular markers to understand signaling pathways?

Integrating ITGB3 data with other molecular markers provides comprehensive insights into signaling networks. When using ITGB3 (Ab-773) Antibody alongside other molecular tools, researchers can implement several integration strategies. Multiplex immunofluorescence combining antibodies against ITGB3 with antibodies targeting its known interaction partners (such as ITGAV, CHL1, or IGF1R) can visualize co-localization patterns and reveal spatial relationships within signaling hubs . Correlation analysis between ITGB3 phosphorylation levels and activation markers of downstream pathways (such as FAK, Src, or PI3K/AKT) can establish signaling hierarchies and feedback mechanisms. For systems-level analysis, protein and phosphoprotein data from antibody-based assays can be integrated with transcriptomic data to identify potential discordances between mRNA and protein levels due to miRNA regulation, as demonstrated for ITGB3 and miR-221/222 . Pathway enrichment analysis using protein interaction databases can position ITGB3 within larger signaling networks, including those related to RAS signaling and apoptotic pathways in which ITGB3 has been implicated . Mathematical modeling approaches, such as Bayesian network analysis or ordinary differential equation models, can integrate quantitative antibody-derived data to predict system behaviors under various perturbations. For clinical samples, correlating ITGB3 levels or phosphorylation status with disease markers can identify potential biomarker applications, particularly in bleeding disorders or conditions where ITGB3 dysregulation has been documented .