Phospho-SNCA (Tyr125) Antibody

What is alpha-synuclein Tyr125 phosphorylation and why is it important in neurodegenerative disease research?

Alpha-synuclein (SNCA) phosphorylation at tyrosine 125 (Tyr125) represents one of several post-translational modifications that occur on this protein, which is centrally involved in Parkinson's disease and other synucleinopathies. While phosphorylation at serine 129 (pS129) has been extensively studied as a primary marker of alpha-synuclein pathology, tyrosine 125 phosphorylation appears to have distinct functional implications in disease pathogenesis . Research suggests that Tyr125 phosphorylation occurs via a PTK2B-dependent pathway during cellular stress conditions, potentially representing a regulatory mechanism in alpha-synuclein aggregation dynamics . Understanding this modification is crucial for deciphering the complex biochemical cascade that leads to alpha-synuclein aggregation and subsequent neurodegeneration in Parkinson's disease and related disorders .

How do antibodies against phospho-SNCA (Tyr125) differ from those targeting other alpha-synuclein phosphorylation sites?

Phospho-SNCA (Tyr125) antibodies specifically recognize alpha-synuclein phosphorylated at tyrosine residue 125, which differentiates them from antibodies targeting other phosphorylation sites such as the more commonly studied pS129. The key differences include:

• Epitope recognition: Phospho-Tyr125 antibodies target a distinct region of the alpha-synuclein protein, typically within the C-terminal domain between amino acids 91-140, compared to pS129 antibodies which recognize the extreme C-terminus .

• Sensitivity to neighboring modifications: Recent research demonstrates that the presence of multiple pathology-associated C-terminal post-translational modifications can differentially influence the detection capacity of phosphorylation site-specific antibodies . Phospho-Tyr125 antibodies may exhibit different detection patterns when neighboring amino acids are modified, compared to pS129 antibodies.

• Cross-reactivity profiles: Each phospho-specific antibody has a unique cross-reactivity profile with other phosphorylated proteins, which necessitates careful validation in experimental applications .

What are the typical applications for Phospho-SNCA (Tyr125) antibodies in neuroscience research?

Phospho-SNCA (Tyr125) antibodies serve multiple essential functions in neuroscience research:

These applications allow researchers to track phosphorylated alpha-synuclein in various experimental contexts, from cellular models to patient-derived samples . The selection of appropriate application and optimization of conditions should be determined based on the specific experimental design and biological questions being addressed.

How can researchers validate the specificity of Phospho-SNCA (Tyr125) antibodies for experimental applications?

Validating Phospho-SNCA (Tyr125) antibody specificity requires a multi-faceted approach:

• Protein standards and controls: Include synthetic phosphorylated peptides corresponding to the region around Tyr125 as positive controls. Use non-phosphorylated alpha-synuclein and alpha-synuclein with phosphorylation at other sites (e.g., pS129) to assess cross-reactivity .

• Genetic controls: Employ alpha-synuclein knockout models (cells or tissues) as negative controls to identify non-specific binding. Recent research has demonstrated that many pS129 antibodies detect non-specific bands in alpha-synuclein knockout samples that could be mistaken for alpha-synuclein species .

• Competition assays: Pre-incubate antibodies with phosphorylated peptides to confirm binding specificity through signal abolishment.

• Phosphatase treatment: Treat samples with phosphatases to remove phosphorylation and confirm loss of antibody binding.

• Multiple antibody validation: Compare results using different Phospho-Tyr125 antibodies from various sources, as demonstrated in systematic studies of pS129 antibodies .

What are the critical factors affecting the detection of phosphorylated alpha-synuclein at Tyr125 in experimental systems?

Several critical factors influence the reliable detection of phosphorylated alpha-synuclein at Tyr125:

• Co-occurring post-translational modifications: Recent research demonstrates that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications differentially influences the detection of phosphorylated alpha-synuclein by specific antibodies . For example, phosphorylation at Tyr125 or truncation at residue 133 or 135 can affect detection of other phosphorylation sites and potentially vice versa.

• Antibody clone and source: Different antibodies (monoclonal vs. polyclonal, different hosts) show varying performance in detecting different forms of phosphorylated alpha-synuclein (monomeric vs. aggregated) .

• Sample preparation methods: Fixation procedures, extraction buffers, and denaturing conditions significantly impact epitope accessibility and antibody binding.

• Alpha-synuclein aggregation state: Monomeric and aggregated forms of phosphorylated alpha-synuclein may be differentially detected by antibodies, with some antibodies performing better for one form over the other .

• Phosphorylation dynamics: The transient nature of Tyr125 phosphorylation and its potential regulation during cellular stress responses require careful consideration of experimental timing and conditions .

Recent systematic assessments suggest that researchers should carefully select antibodies based on their specific experimental needs and thoroughly validate them in their particular experimental systems to ensure accurate detection of phosphorylated alpha-synuclein species .

What are the optimal sample preparation methods for Phospho-SNCA (Tyr125) antibody applications?

Optimal sample preparation for Phospho-SNCA (Tyr125) antibody applications varies by technique:

For Western Blotting:

• Use phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in lysis buffers to preserve phosphorylation status.

• Prepare samples in denaturing conditions (SDS) to ensure epitope accessibility.

• Include both reducing and non-reducing conditions in validation experiments, as disulfide bonds may affect epitope accessibility.

For Immunohistochemistry/Immunofluorescence:

• Optimize fixation protocols—paraformaldehyde (4%) is commonly used for alpha-synuclein studies.

• Consider antigen retrieval methods (heat-induced or enzymatic) to improve epitope accessibility.

• Block endogenous peroxidase activity when using HRP-conjugated detection systems.

For Cell-Based Assays:

• Consider permeabilization methods that preserve cellular architecture while allowing antibody access.

• Use physiologically relevant models expressing alpha-synuclein at endogenous levels when possible.

• Include treatments that modulate phosphorylation (kinase activators/inhibitors) as controls.

Recent research emphasizes the importance of using appropriate protein standards and controls when investigating alpha-synuclein under physiological conditions, as the specificity of antibody detection can be significantly affected by sample preparation methods .

How can researchers distinguish between true Tyr125 phosphorylation signals and experimental artifacts?

Distinguishing true phosphorylation signals from artifacts requires a comprehensive validation approach:

• Genetic controls: Utilize alpha-synuclein knockout models as negative controls in all experiments. Research has shown that many phospho-specific antibodies detect non-specific bands in alpha-synuclein knockout samples that could be mistaken for alpha-synuclein species .

• Phosphatase treatments: Treat duplicate samples with lambda phosphatase or tyrosine-specific phosphatases to demonstrate phosphorylation-dependent signaling.

• Peptide competition: Pre-absorb antibodies with phosphorylated and non-phosphorylated peptides to confirm signal specificity.

• Multiple antibody confirmation: Use at least two different antibodies targeting the same phosphorylation site but raised against different epitopes or from different sources.

• Molecular weight verification: Carefully analyze the molecular weight of detected bands—true alpha-synuclein monomers typically run at ~14-17 kDa, while higher molecular weight species may represent aggregates or cross-reactive proteins.

• Mass spectrometry validation: When possible, confirm phosphorylation sites through mass spectrometry analysis of immunoprecipitated samples.

Research has demonstrated that not all phospho-specific antibodies capture the biochemical and morphological diversity of alpha-synuclein pathology, emphasizing the need for rigorous validation approaches to avoid misinterpretation of experimental results .

What are the considerations for using Phospho-SNCA (Tyr125) antibodies in combination with other alpha-synuclein antibodies?

When combining Phospho-SNCA (Tyr125) antibodies with other alpha-synuclein antibodies, researchers should consider several important factors:

• Epitope compatibility: Ensure that antibodies targeting different regions or modifications of alpha-synuclein do not compete for binding sites or sterically hinder each other's access when used in combination.

• Host species selection: Select primary antibodies raised in different host species (e.g., rabbit anti-pTyr125 with mouse anti-total alpha-synuclein) to enable simultaneous detection with species-specific secondary antibodies in co-labeling experiments.

• Validation of combination protocols: Validate each antibody individually before combining them, and then verify that their combined use does not alter detection patterns.

• Cross-reactivity assessment: Assess potential cross-reactivity between secondary antibodies and non-targeted primary antibodies in multiplexed detection systems.

• Influence of neighboring modifications: Be aware that modifications at one site may affect antibody binding to neighboring sites. Recent research demonstrates that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of phosphorylated species by specific antibodies .

• Quantification considerations: When quantifying signals from multiple antibodies, account for potential differences in antibody affinity and detection sensitivity by using appropriate normalization methods.

How should researchers interpret conflicting results from different Phospho-SNCA (Tyr125) antibody sources?

Interpreting conflicting results from different Phospho-SNCA (Tyr125) antibodies requires a systematic approach:

• Antibody characterization comparison: Review the specific epitopes recognized by each antibody. Those generated against slightly different immunogens (e.g., different peptide lengths around Tyr125) may yield different results .

• Specificity evaluation: Assess each antibody's validation data, particularly regarding specificity for phosphorylated versus non-phosphorylated forms and potential cross-reactivity with other phosphorylated proteins.

• Consideration of post-translational modification interplay: Recent research has demonstrated that neighboring post-translational modifications can significantly affect antibody binding. The presence of other modifications near Tyr125 may influence detection by different antibodies to varying degrees .

• Experimental system differences: Evaluate whether conflicting results may be due to differences in experimental systems (cell types, tissue processing, detection methods) rather than antibody performance.

• Consensus approach: When possible, rely on results that are consistent across multiple antibodies and experimental approaches rather than data from a single antibody.

Recent systematic studies of phospho-specific alpha-synuclein antibodies have identified significant variations in antibody performance across different experimental systems, with some antibodies showing insensitivity to neighboring post-translational modifications while others are significantly affected . These findings underscore the importance of thorough antibody validation and careful interpretation of potentially conflicting results.

What are common pitfalls in experimental design when using Phospho-SNCA (Tyr125) antibodies?

Researchers should be aware of several common pitfalls when designing experiments with Phospho-SNCA (Tyr125) antibodies:

How can researchers address non-specific binding issues with Phospho-SNCA (Tyr125) antibodies?

Addressing non-specific binding issues with Phospho-SNCA (Tyr125) antibodies requires a multi-faceted approach:

• Optimization of blocking conditions: Systematically test different blocking agents (BSA, normal serum, commercial blocking buffers) and concentrations to minimize non-specific binding.

• Antibody titration: Perform careful antibody dilution series to identify the optimal concentration that maximizes specific signal while minimizing background.

• Stringent washing protocols: Implement more rigorous washing steps (increased duration, wash buffer optimization) to remove weakly bound antibodies.

• Pre-absorption strategies: Pre-incubate antibodies with non-specific proteins or with phosphorylated peptides corresponding to potential cross-reactive epitopes to reduce non-specific binding.

• Alternative detection systems: Compare different detection systems (fluorescent vs. chromogenic) and amplification methods to identify approaches that maximize signal-to-noise ratio.

• Sample preparation refinement: Modify fixation, permeabilization, or extraction protocols to better preserve specific epitopes while reducing exposure of non-specific binding sites.

• Knockout validation: Always include alpha-synuclein knockout samples as controls to identify non-specific bands. Research has shown that many phospho-specific antibodies detect non-specific bands in alpha-synuclein knockout samples that could be mistaken for alpha-synuclein species .

Systematic studies have demonstrated that most phospho-specific alpha-synuclein antibodies show some degree of cross-reactivity, emphasizing the importance of thorough validation and optimization to distinguish specific from non-specific signals in experimental applications .

How can Phospho-SNCA (Tyr125) antibodies be utilized in alpha-synuclein seeding and propagation models?

Phospho-SNCA (Tyr125) antibodies offer valuable tools for investigating alpha-synuclein seeding and propagation:

• Tracking phosphorylation changes during seeding: Monitor the temporal and spatial phosphorylation patterns at Tyr125 following introduction of alpha-synuclein seeds in cellular or animal models, potentially revealing insights into the relationship between phosphorylation and aggregation dynamics.

• Investigating strain-specific phosphorylation: Compare Tyr125 phosphorylation patterns across different alpha-synuclein pathological strains to determine whether this modification contributes to strain-specific properties.

• Assessing phosphorylation in recipient cells: In cell-to-cell transmission models, examine whether phosphorylation status changes during transmission or whether specific phosphorylated species are preferentially transmitted.

• Immunodepletion experiments: Selectively deplete phosphorylated species using Phospho-SNCA (Tyr125) antibodies to assess their specific contribution to seeding activity in propagation assays.

• Cross-validation in seeding models: Use multiple phospho-specific antibodies targeting different sites (including Tyr125) to comprehensively characterize the phosphorylation landscape during seeding and propagation.

Research has demonstrated that phospho-specific antibodies can be valuable tools in seeding models of alpha-synuclein pathology formation, though their performance may vary across different experimental systems . Recent systematic assessments suggest that careful antibody selection and validation are essential when applying these tools to complex seeding and propagation models.

What is the relationship between Tyr125 and Ser129 phosphorylation in alpha-synuclein pathology?

The relationship between Tyr125 and Ser129 phosphorylation in alpha-synuclein pathology remains an area of active research:

• Differential occurrence in pathology: While phosphorylation at Ser129 is extensively documented in Lewy bodies and other alpha-synuclein pathological aggregates (with approximately 90% of aggregated alpha-synuclein being phosphorylated at this site), the prevalence and significance of Tyr125 phosphorylation in pathological contexts requires further characterization .

• Functional interplay: Evidence suggests these phosphorylation events may have opposing effects—some studies indicate that Tyr125 phosphorylation may be neuroprotective and is reduced in disease states, while Ser129 phosphorylation increases in pathological conditions.

• Sequential phosphorylation dynamics: Current research has not fully elucidated whether phosphorylation at one site influences the likelihood or kinetics of phosphorylation at the other site, though the proximity of these residues in the C-terminal region suggests potential interactions.

• Detection challenges: The co-occurrence of phosphorylation at multiple sites can affect antibody detection, potentially leading to underestimation of doubly phosphorylated species in experimental systems .

• Kinase regulation: Different kinases are responsible for phosphorylation at these sites (Tyr125 via PTK2B-dependent pathways and Ser129 via multiple kinases including CK1 and PLKs), suggesting distinct regulatory mechanisms and potentially different triggers for each modification .

Recent research emphasizes the importance of considering the interplay between multiple post-translational modifications when investigating alpha-synuclein pathology, as these modifications may collectively influence alpha-synuclein aggregation, toxicity, and clearance mechanisms .

How can researchers develop improved detection methods for phosphorylated alpha-synuclein species in complex biological samples?

Developing improved detection methods for phosphorylated alpha-synuclein requires innovative approaches:

• Next-generation antibody engineering: Engineer antibodies with enhanced specificity for phosphorylated epitopes that are insensitive to neighboring post-translational modifications, addressing a key limitation identified in recent systematic studies .

• Multiplexed detection systems: Develop assays that simultaneously detect multiple phosphorylation sites and other post-translational modifications to provide a comprehensive profile of alpha-synuclein modification states.

• Conformation-specific detection: Create tools that specifically recognize phosphorylated alpha-synuclein in particular conformational states (monomeric vs. oligomeric vs. fibrillar) to better characterize the relationship between phosphorylation and aggregation.

• Mass spectrometry integration: Combine antibody-based enrichment with mass spectrometry analysis to precisely quantify phosphorylated species and identify co-occurring modifications.

• Proximity ligation approaches: Implement proximity ligation assays to detect specific combinations of modifications or to visualize interactions between phosphorylated alpha-synuclein and other proteins in situ.

• Single-molecule techniques: Apply single-molecule detection methods to characterize the heterogeneity of phosphorylation patterns within alpha-synuclein populations.

• Biosensor development: Create biosensors that can specifically detect phosphorylated alpha-synuclein species in living cells or in real-time from biological fluids.

Recent research underscores the need for more phospho-specific antibodies that are not sensitive to neighboring post-translational modifications and more thorough characterization and validation of existing and new antibodies . These improvements will be critical for advancing our understanding of the complex role of alpha-synuclein phosphorylation in neurodegenerative disease pathogenesis.