Phospho-SNCA (S129) Recombinant Monoclonal Antibody

What is alpha-synuclein phosphorylation at S129 and why is it important in neurodegenerative research?

Alpha-synuclein (SNCA) phosphorylation at serine 129 (pS129) represents a critical post-translational modification substantially increased in Lewy body diseases such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). This modification is found in approximately 90% of alpha-synuclein in Lewy bodies compared to only about 4% in normal brain tissue . The importance of pS129-SNCA stems from its role as the primary biomarker for tracking alpha-synuclein pathology in both brain and peripheral tissues of patients with synucleinopathies .

Alpha-synuclein normally functions as a neuronal protein involved in multiple aspects of synaptic activity, including:

• Regulation of synaptic vesicle trafficking and neurotransmitter release

• Enhancement of vesicle priming, fusion, and dilation of exocytotic fusion pores

• Molecular chaperoning of synaptic fusion components (SNAREs)

• Modulation of dopamine transporter (DAT1) activity

What experimental techniques can Phospho-SNCA (S129) antibodies be used for?

Phospho-SNCA (S129) antibodies have been validated for multiple experimental applications:

Methodology note: When performing western blots, 1% SDS hot lysate method is recommended to reduce detection of oligomers compared to the RIPA method, which shows stronger detection of high molecular weight species around 100 kDa .

How should Phospho-SNCA (S129) antibodies be stored and handled for optimal performance?

For optimal performance of Phospho-SNCA (S129) antibodies:

• Long-term storage: Store at -10°C to -25°C or -20°C for up to one year

• Short-term storage: For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as this may compromise antibody integrity

• Typical antibody formulations include:

  • Buffer solutions containing PBS with 0.02% sodium azide

  • 50% glycerol, pH 7.2 for stability

  • Concentrations ranging from 1-3 mg/ml depending on manufacturer

How do neighboring post-translational modifications affect the specificity of Phospho-SNCA (S129) antibodies?

Recent research has revealed critical considerations regarding antibody specificity:

The presence of multiple pathology-associated C-terminal post-translational modifications (PTMs) significantly influences the detection capabilities of pS129-SNCA antibodies. Specifically:

• Phosphorylation at tyrosine 125 (pY125) can interfere with pS129 detection by some antibodies

• C-terminal truncations at residues 133 or 135 affect antibody binding differently depending on epitope location

• Antibodies targeting residues 115-122 show weaker signals when alpha-synuclein is truncated at residue 120

A systematic assessment revealed that only two commonly used pS129 antibodies were found to be completely insensitive to neighboring PTMs, making epitope characterization crucial before experimental design .

These findings underscore the importance of thorough antibody validation, as not all pS129 antibodies can capture the biochemical and morphological diversity of alpha-synuclein pathology .

What are the potential cross-reactivity concerns with Phospho-SNCA (S129) antibodies, and how can they be mitigated?

Despite manufacturer claims of specificity, several studies have demonstrated unexpected cross-reactivity issues with pS129-SNCA antibodies:

Most pS129 antibodies showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in alpha-synuclein knockout samples that could be easily mistaken for monomeric or high molecular weight alpha-synuclein species .

To mitigate these concerns:

• Always use appropriate controls:

  • Alpha-synuclein knockout samples as negative controls

  • Recombinant phosphorylated and non-phosphorylated proteins as standards

  • Alkaline phosphatase treatment of samples to verify phosphorylation-specific signals

• Validate with multiple antibodies targeting different epitopes:

  • Combine N-terminal, NAC region, and C-terminal antibodies

  • Use both phosphorylation-dependent and independent antibodies

• Implement specialized blocking procedures:

  • 5% non-fat dry milk (NFDM) in TBST has been shown effective for minimizing background

What is the temporal relationship between alpha-synuclein aggregation and S129 phosphorylation in disease progression?

Recent research has clarified the timing of pS129 modification during disease progression:

Studies using an antibody (4B1) specifically recognizing non-phosphorylated S129-alpha-synuclein demonstrated that:

• S129 residue is more efficiently phosphorylated when the protein is already aggregated

• In mouse models, aggregation of non-phosphorylated alpha-synuclein precedes pS129-alpha-synuclein formation

Time-course experiments in:

• Organotypic mouse hippocampal cultures

• Mice injected with alpha-synuclein preformed fibrils

Both revealed that aggregation of non-phosphorylated alpha-synuclein occurs first, followed by the appearance of pS129-alpha-synuclein .

In human postmortem brain tissue from PD and DLB patients, an inverse relationship exists between relative abundance of non-phosphorylated alpha-synuclein and disease duration, suggesting progressive phosphorylation of aggregated species over time .

How does phosphorylation at S129 affect alpha-synuclein aggregation properties and cytotoxicity?

Contrary to earlier assumptions, pS129 modification may actually inhibit rather than promote alpha-synuclein aggregation:

Experimental evidence demonstrates that:

• pS129-alpha-synuclein inhibits fibril formation in vitro

• In samples containing 50% and 100% pS129-alpha-synuclein, fibril formation was almost completely inhibited (Thioflavin-S counts ~2,000 after 20 days) compared to non-phosphorylated samples (~35,000 Thioflavin-S counts)

• Even 20% pS129-alpha-synuclein content resulted in >50% reduction in aggregation propensity

Regarding cytotoxicity:

• pS129-alpha-synuclein demonstrated reduced seeding and aggregation propensity in neuroblastoma cell models

• Molecular simulations suggest pS129 may stabilize alpha-synuclein monomers, resisting further aggregation

• pS129-alpha-synuclein seeding showed no effect on the viability of neuroblastoma cells, in contrast to wild-type alpha-synuclein

These findings suggest a potential protective role for pS129-alpha-synuclein, occurring subsequent to initial protein aggregation and apparently inhibiting further aggregation .

What are the optimal protocols for detecting phospho-S129 alpha-synuclein in brain tissue samples?

For reliable detection of pS129-alpha-synuclein in brain tissue:

Immunohistochemistry protocol:

• Tissue preparation:

  • Fix tissue in formalin and embed in paraffin

  • Section at 5-10 μm thickness

• Antigen retrieval:

  • Heat-mediated antigen retrieval with citrate buffer (pH 6.0)

  • 20 minutes incubation time is optimal for most samples

• Antibody incubation:

  • Primary antibody dilution: 1:50-1:200 (optimize for each antibody)

  • Incubation time: 15 hours at 4°C or 15 minutes at room temperature

  • Secondary detection: HRP-conjugated compact polymer system or biotinylated secondary antibody (1:200)

• Visualization:

  • DAB as chromogen for brightfield microscopy

  • Counterstain with hematoxylin for structural context

Western blot optimization:

• Sample preparation: 1% SDS hot lysate method reduces detection of oligomers compared to RIPA

• Blocking: 5% non-fat dry milk in TBST is effective

• Primary antibody: 1:1000 dilution is typically optimal

• Expected bands: 14-18 kDa (monomeric) and ~100 kDa (oligomeric)

• Controls: Include alkaline phosphatase-treated samples to confirm phospho-specificity

How can researchers differentiate between physiological and pathological pS129-alpha-synuclein?

Distinguishing physiological from pathological pS129-alpha-synuclein requires multiple analytical approaches:

• Morphological characteristics:

  • Pathological pS129-alpha-synuclein typically forms punctate or fibrillar inclusions

  • Physiological pS129-alpha-synuclein shows diffuse cytoplasmic staining

• Solubility-based fractionation:

  • Pathological forms are enriched in detergent-insoluble fractions

  • Sequential extraction with increasingly harsh detergents:

    1. High-salt buffer (soluble fraction)

    2. 1% Triton X-100 (membrane-associated fraction)

    3. 1% sarkosyl (aggregated fraction)

• Co-localization with pathological markers:

  • Pathological pS129-alpha-synuclein co-localizes with ubiquitin and p62

  • Using antibodies that differentiate non-phosphorylated from phosphorylated S129 (such as 4B1)

• Seeding activity assessment:

  • Real-time quaking-induced conversion (RT-QuIC) assay shows differential seeding between physiological and pathological forms

  • Pathological forms show higher seeding efficiency

What control samples should be used to validate experimental findings with pS129-SNCA antibodies?

Rigorous experimental design requires appropriate controls:

Essential negative controls:

• Alpha-synuclein knockout tissue/cells to verify antibody specificity

• Secondary antibody-only controls to assess non-specific binding

Positive controls:

• Recombinant pS129-alpha-synuclein at known concentrations

• Brain samples from confirmed PD/DLB cases (particularly substantia nigra)

Specificity controls:

• Alkaline phosphatase treatment of samples to remove phosphorylation

• Dot blot analysis comparing pS129 peptide vs. unmodified peptide

• Competitive blocking with immunizing peptide

Parallel antibody validation:

• Use multiple antibodies targeting different epitopes

• Compare results between phosphorylation-dependent and independent antibodies

How should researchers address the heterogeneity of post-translational modifications in alpha-synuclein pathology?

To comprehensively characterize the complex landscape of alpha-synuclein modifications:

• Develop a comprehensive antibody panel targeting:

  • Different alpha-synuclein regions (N-terminal, NAC, C-terminal)

  • Various PTMs (phosphorylation, nitration, truncation)

  • Conformational epitopes specific to aggregated forms

• Sequential tissue staining approach:

  • Apply multiple rounds of immunostaining to the same tissue section

  • Strip and re-probe with different antibodies

  • Use spectral unmixing to differentiate signals from multiple fluorophores

• Mass spectrometry characterization:

  • Apply enrichment strategies to isolate specific PTM-containing fragments

  • Quantify relative abundance of different modifications

  • Map the distribution of modifications across the protein sequence

Recent research has revealed distinct and heterogeneously modified alpha-synuclein pathologies rich in:

• pS129

• Tyrosine 39 (Y39) nitration

• N- and C-terminal tyrosine phosphorylations

These modifications occur with different distributions in neurons and glia, requiring careful selection of antibodies to capture the full spectrum of pathology .

How can researchers utilize Phospho-SNCA (S129) antibodies in longitudinal studies of disease progression?

Implementing pS129-SNCA antibodies in longitudinal studies requires strategic approaches:

• Staged tissue collection from animal models:

  • Collect tissues at defined timepoints after alpha-synuclein fibril injection

  • Track the spatiotemporal spread of pathology

  • Compare timing of non-phosphorylated vs. phosphorylated alpha-synuclein appearance

• Quantitative assessment methods:

  • Develop standardized scoring systems for pathology burden

  • Use digital image analysis for objective quantification

  • Apply machine learning algorithms to identify subtle pathological patterns

• Correlation with functional outcomes:

  • Pair immunohistochemical analysis with behavioral/motor assessments

  • Establish relationships between regional pathology and specific symptoms

  • Determine whether pS129-SNCA burden predicts functional decline rate

Research using 4B1 antibody (specific for non-phosphorylated S129) revealed:

• In postmortem brain tissue from PD and DLB patients, an inverse relationship exists between relative abundance of non-phosphorylated alpha-synuclein and disease duration

• This suggests progressive phosphorylation of aggregated species over time and potential use as a disease progression marker

What are the implications of pS129-SNCA's inhibitory effect on aggregation for therapeutic development?

The discovery that pS129 modification inhibits rather than promotes alpha-synuclein aggregation has significant implications:

• Reassessment of therapeutic strategies:

  • Previous approaches targeting reduction of pS129-SNCA may need reconsideration

  • Kinase inhibitors targeting S129 phosphorylation could potentially worsen pathology

  • Enhancing specific phosphorylation pathways might represent a novel therapeutic avenue

• Disease monitoring considerations:

  • Reduction in pS129-SNCA may not necessarily indicate therapeutic efficacy

  • The ratio of phosphorylated to non-phosphorylated alpha-synuclein may be more informative than absolute levels

• Mechanistic explorations:

  • Molecular simulations suggest pS129 stabilizes alpha-synuclein monomers

  • Understanding how this stabilization occurs could inform structure-based drug design

  • Developing compounds that mimic the structural effects of phosphorylation without requiring enzymatic modification

The finding that pS129-alpha-synuclein occurs subsequent to initial protein aggregation and inhibits further aggregation suggests a potential protective role, which has major implications for understanding pathobiology and clinical trial endpoints .

What technical challenges exist in developing antibodies that can distinguish oligomeric vs. fibrillar forms of pS129-SNCA?

Developing conformation-specific antibodies for phosphorylated alpha-synuclein presents unique challenges:

• Epitope accessibility issues:

  • The S129 residue may be differentially exposed in various aggregate conformations

  • Protein folding patterns in oligomers vs. fibrils create distinct epitope landscapes

  • Antibody size limitations may prevent access to buried epitopes

• Stability considerations:

  • Alpha-synuclein oligomers are transient and heterogeneous

  • Preserving native conformations during sample processing is difficult

  • Different buffer conditions can alter conformational states

• Technical approaches to address these challenges:

  • Phage display selection using conformationally-stabilized preparations

  • Competitive selection strategies to eliminate cross-reactive antibodies

  • Single-domain antibody development for better access to buried epitopes

  • Conformation-specific immunization protocols using stabilized oligomers or fibrils

Current research indicates that using antibodies recognizing different epitopes can help distinguish various aggregation states, as high molecular weight bands around 100 kDa are typically associated with oligomeric forms, while specific fibril-associated epitopes may be masked in certain aggregation states .

How do technical variations in sample preparation affect the detection of pS129-SNCA in experimental models and human tissues?

Sample preparation methodology significantly impacts pS129-SNCA detection and quantification:

• Tissue preservation effects:

• Lysate preparation variables:

  • RIPA buffer enhances detection of high-molecular-weight species (~100 kDa)

  • 1% SDS hot lysate method reduces oligomer detection

  • Phosphatase inhibitors are critical to prevent ex vivo dephosphorylation

• Antigen retrieval optimization:

  • Heat-mediated retrieval with citrate buffer (pH 6.0) is generally effective

  • Extended retrieval times (>20 min) may reduce signal for some antibodies

  • Formic acid pretreatment can enhance detection of aggregated forms

• Validation recommendations:

  • Test multiple fixation and extraction protocols with each antibody

  • Include phosphatase-treated controls to confirm phospho-specificity

  • Compare detection methods (IHC vs. WB vs. ELISA) to confirm findings

  • Use standardized protocols when comparing across experimental conditions or patient cohorts