Phospho-SRC (Tyr529) Antibody

What is the biological significance of SRC phosphorylation at Tyrosine 529?

Phosphorylation of SRC at Tyrosine 529 (Tyr529) represents a critical inhibitory modification that maintains SRC in an inactive conformation. When phosphorylated at this residue, SRC adopts a closed configuration where the SH2 domain engages with Tyr529, while the SH3 domain interacts with the SH2-kinase linker region. This conformation prevents the autophosphorylation of Tyr419, thereby suppressing SRC kinase activity .

In cellular contexts, the phosphorylation status of SRC Tyr529 serves as a molecular switch that regulates numerous signaling pathways involved in cell adhesion, migration, proliferation, and differentiation. The inhibitory phosphorylation is typically maintained by C-terminal SRC kinase (CSK), while dephosphorylation by protein tyrosine phosphatases (including PTPα) releases this inhibition .

How do different cellular conditions affect SRC Tyr529 phosphorylation levels?

Cellular adhesion conditions significantly impact SRC Tyr529 phosphorylation. Research demonstrates that cells plated on fibronectin (which facilitates focal adhesion formation) exhibit markedly lower levels of inhibitory Tyr529 phosphorylation compared to cells plated on poly-L-lysine (where focal adhesions are absent) . This observation aligns with the understanding that SRC activation via Tyr529 dephosphorylation occurs during adhesion and spreading on fibronectin substrates.

Cytokine stimulation also modulates SRC Tyr529 phosphorylation. For instance, IL-1 treatment induces a modest increase in Tyr529 phosphorylation after 15 minutes, though this effect is not evident at earlier time points . The time-dependent nature of these modifications suggests complex regulatory dynamics involving multiple signaling pathways.

What is the relationship between Tyr529 phosphorylation and other SRC regulatory sites?

The regulation of SRC involves coordinated modifications at multiple sites, with Tyr529 and Tyr419 serving as the primary regulatory phosphorylation sites. These sites exhibit an inverse relationship:

When Tyr529 is dephosphorylated, the intramolecular interaction between the SH2 domain and Tyr529 is disrupted, allowing Tyr419 to become autophosphorylated. This sequence of events results in SRC activation . Understanding this interplay is crucial for interpreting experimental results involving SRC activity measurements.

What are the optimal applications for Phospho-SRC (Tyr529) antibodies?

Phospho-SRC (Tyr529) antibodies have been validated for multiple experimental applications, with varying dilution requirements:

When selecting an application, researchers should consider that Western blotting offers quantitative assessment of phosphorylation levels across cell populations, while immunostaining techniques (IHC/IF) provide spatial information regarding the subcellular localization of phosphorylated SRC .

How should researchers optimize Western blot protocols for detecting phospho-SRC (Tyr529)?

For optimal detection of phospho-SRC (Tyr529) by Western blotting, researchers should implement the following protocol refinements:

• Sample preparation: Rapid lysis in the presence of phosphatase inhibitors is critical to preserve the phosphorylation state of Tyr529. Use ice-cold buffers containing sodium orthovanadate, sodium fluoride, and phosphatase inhibitor cocktails.

• Gel electrophoresis: Use 8-10% SDS-PAGE gels for optimal resolution of SRC (approximately 60 kDa).

• Transfer conditions: For efficient transfer of proteins in this molecular weight range, semi-dry transfer (15-20V for 30-40 minutes) or wet transfer (100V for 60-90 minutes) are both suitable.

• Blocking: 5% BSA in TBST is preferable to milk-based blocking solutions, as milk contains phosphoproteins that may interfere with phospho-specific antibody binding.

• Primary antibody incubation: Use the phospho-SRC (Tyr529) antibody at a 1:1000 dilution in 5% BSA/TBST, and incubate overnight at 4°C for optimal signal-to-noise ratio .

• Expected molecular weight: Look for specific bands at approximately 60 kDa (the expected molecular weight for SRC) .

What controls should be incorporated when using phospho-SRC (Tyr529) antibodies?

Rigorous experimental design requires appropriate controls to validate phospho-SRC (Tyr529) antibody specificity:

• Positive controls: Lysates from cells treated with agents known to induce Tyr529 phosphorylation (e.g., cells in suspension or treated with specific kinase activators).

• Negative controls:

  • Lysates from cells treated with SRC family kinase inhibitors

  • Lysates from cells where protein tyrosine phosphatase alpha (PTPα) is overexpressed, leading to Tyr529 dephosphorylation

  • Comparison with PTPα-knockout cells, which typically show elevated Tyr529 phosphorylation

• Peptide competition assay: Pre-incubation of the antibody with the phosphopeptide immunogen should abolish specific signal.

• Phosphatase treatment control: Treating one sample aliquot with lambda phosphatase prior to immunoblotting should eliminate phospho-specific signal .

How can researchers integrate phospho-SRC (Tyr529) analysis into focal adhesion studies?

Focal adhesions represent critical sites of SRC activity regulation. When designing experiments to study SRC phosphorylation at Tyr529 in the context of focal adhesions:

• Substrate selection: Compare cells grown on extracellular matrix proteins that promote focal adhesion formation (fibronectin, collagen) versus non-permissive substrates (poly-L-lysine). Research shows significant differences in Tyr529 phosphorylation levels between these conditions .

• Co-localization studies: Perform immunofluorescence staining for phospho-SRC (Tyr529) alongside focal adhesion markers (paxillin, vinculin) to assess spatial relationships.

• Temporal dynamics: Implement time-course studies during cell adhesion and spreading to capture the dynamic regulation of Tyr529 phosphorylation.

• Integrin manipulation: Use function-blocking antibodies against specific integrins or integrin-activating compounds to assess their impact on SRC Tyr529 phosphorylation.

• Mechanical stimulation: Apply defined mechanical forces to focal adhesions (through substrate stretching or magnetic bead pulling) and monitor changes in Tyr529 phosphorylation status .

What are the challenges in interpreting phospho-SRC (Tyr529) data in complex tissue samples?

Analyzing phospho-SRC (Tyr529) in tissues presents several challenges that require methodological considerations:

• Cell type heterogeneity: Tissues contain multiple cell types with potentially different baseline levels of SRC expression and phosphorylation. Use dual immunofluorescence with cell-type-specific markers to resolve cell-specific patterns.

• Phosphorylation preservation: Rapid tissue fixation is essential to maintain phosphorylation status. For optimal results, tissues should be fixed within minutes of collection using phosphatase inhibitor-supplemented fixatives.

• Epitope masking: Formalin fixation can mask phosphoepitopes. Implement appropriate antigen retrieval methods, with citrate buffer (pH 6.0) heat-induced epitope retrieval showing good results for phospho-SRC detection .

• Quantification challenges: For quantitative analysis in immunohistochemistry, establish standardized scoring systems based on both staining intensity and percentage of positive cells.

• Background signals: Non-specific binding can be problematic in tissue sections. Validate specificity using peptide competition controls and phosphatase-treated serial sections .

How does PTPα-mediated dephosphorylation of SRC Tyr529 coordinate with focal adhesion dynamics?

Protein tyrosine phosphatase alpha (PTPα) plays a critical role in SRC activation through dephosphorylation of the inhibitory Tyr529 residue. Research findings demonstrate:

What methodological approaches can resolve the specificity challenges when studying phospho-SRC (Tyr529) in the context of SRC family kinases?

SRC family kinases (SFKs) share significant sequence homology, particularly around regulatory phosphorylation sites, creating potential cross-reactivity issues with phospho-specific antibodies. To address these challenges:

• Antibody validation: Perform comprehensive validation using cells overexpressing individual SFK members versus SRC-specific knockdown/knockout models.

• Immunoprecipitation-based approach: Use SRC-specific antibodies for immunoprecipitation followed by immunoblotting with phospho-Tyr529 antibodies to confirm the identity of the phosphoprotein.

• Mass spectrometry validation: Apply phosphoproteomic approaches to unambiguously identify the SFK member and its phosphorylation site.

• Functional correlation: Correlate phospho-Tyr529 levels with known SRC-specific substrates to distinguish from other SFK activities.

• SFK isoform-specific inhibitors: Use selective inhibitors when available to dissect the contribution of individual SFK members to the observed phospho-signal .

How can phospho-SRC (Tyr529) antibodies be integrated into multiplexed detection systems?

Modern research increasingly requires simultaneous detection of multiple phosphorylation sites to understand signaling network dynamics. For multiplexed analysis of SRC phosphorylation:

What factors contribute to inconsistent phospho-SRC (Tyr529) detection in Western blotting?

Researchers frequently encounter variability in phospho-SRC (Tyr529) signal intensity. Common causes and solutions include:

• Rapid dephosphorylation during sample preparation: Ensure immediate addition of phosphatase inhibitors (10 mM sodium orthovanadate, 50 mM sodium fluoride) to lysis buffers and maintain samples at 4°C throughout processing.

• Insufficient blocking: Extend blocking time to 2 hours at room temperature using 5% BSA in TBST rather than milk-based blockers.

• Antibody specificity variations: Different commercial antibodies may recognize slightly different epitopes around Tyr529. Validate using phosphopeptide competition assays and test multiple antibodies if possible.

• Gel percentage considerations: Using 8% rather than 10% or 12% gels may improve resolution of phospho-SRC bands around the 60 kDa range.

• Transfer efficiency issues: For phosphoproteins, wet transfer systems often provide more consistent results than semi-dry systems. Consider using PVDF membranes rather than nitrocellulose for improved protein retention .

How should researchers approach conflicting results between phospho-SRC (Tyr529) levels and functional SRC activity?

Discrepancies between phospho-Tyr529 detection and functional SRC activity measures can arise from several factors:

• Multiple regulatory sites: While Tyr529 phosphorylation is inhibitory, SRC activity is also regulated by phosphorylation at Tyr419 and other sites. Always measure both regulatory phosphorylation sites simultaneously.

• Stoichiometry considerations: Even partial dephosphorylation of Tyr529 across a population of SRC molecules may be sufficient to generate significant kinase activity.

• Localization effects: Total cellular phospho-Tyr529 levels may not reflect changes in specific subcellular compartments where SRC is being activated. Consider fractionation approaches to resolve this issue.

• Timing discrepancies: Phosphorylation changes may precede detectable changes in downstream substrate phosphorylation. Implement detailed time-course experiments to capture these dynamics.

• Validation approaches: Complement phospho-specific immunoblotting with direct SRC kinase activity assays using specific substrates to resolve apparent contradictions .