Phospho-MAPT (S519) Antibody

What is the target of Phospho-MAPT (S519) Antibody and why is it significant?

Phospho-MAPT (S519) Antibody specifically recognizes the microtubule-associated protein tau (MAPT) when phosphorylated at serine 519. This phosphorylation site is significant in neurodegenerative tauopathies, where tau protein becomes hyperphosphorylated and forms pathological inclusions . The antibody allows researchers to specifically detect and study this post-translational modification, which contributes to tau aggregation and neurofibrillary tangle formation.

Tau protein has six isoforms with two 3' UTR variants in humans, and phosphorylation at specific sites like S519 affects its normal function in stabilizing microtubules . The ability to specifically detect phosphorylation at S519 provides valuable insights into disease progression mechanisms.

What applications are Phospho-MAPT (S519) Antibody suitable for?

Based on validated testing, Phospho-MAPT (S519) Antibody is suitable for the following applications:

The antibody has been validated for specificity in detecting endogenous levels of tau protein only when phosphorylated at serine 519 . Some versions may also be suitable for immunohistochemistry applications, particularly in examining pathological inclusions in human brain tissue and transgenic mouse models of tauopathy .

How should Phospho-MAPT (S519) Antibody be stored and handled?

For optimal performance and longevity:

• Store the antibody at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles as this can degrade antibody quality

• For short-term storage and frequent use, storing at 4°C for up to one month is acceptable

• The antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide as a preservative

How can I validate the specificity of Phospho-MAPT (S519) Antibody in my experimental system?

Validating phospho-specific antibodies requires multiple approaches:

• Phosphopeptide/non-phosphopeptide competition experiments: Incubate the antibody with the phosphopeptide corresponding to the target site before using it in your application. The signal should be blocked by the phosphopeptide but not by the non-phosphorylated peptide .

• Analysis of site-directed mutants: Use samples where the serine at position 519 has been mutated to alanine (S519A). The antibody should not recognize this mutant form, confirming its phospho-specificity .

• Dephosphorylation assay: Treat your samples with phosphatase before detection. The signal should be significantly reduced or eliminated after phosphatase treatment .

• Multiple cell line validation: Test the antibody in multiple cell lines with known tau expression profiles and phosphorylation states to establish consistent performance .

As noted in recent structural studies of phospho-specific antibodies: "The fact that these antibodies were raised using phosphorylated peptides as antigens illustrates the importance of performing negative selections to remove nonselective binders during the antibody screening process" .

What are the optimal conditions for using Phospho-MAPT (S519) Antibody in Western blot applications?

For optimal Western blot results:

• Sample preparation: Use fresh tissue lysates or cultured cells treated with phosphatase inhibitors immediately upon collection to preserve phosphorylation status

• Protein loading: Load 20-50 μg of total protein per lane

• Blocking: Use 5% BSA in TBST rather than milk (which contains phosphatases that may reduce signal)

• Antibody dilution: Start with 1:1000 dilution in 5% BSA/TBST

• Incubation: Overnight at 4°C for primary antibody

• Detection: Use highly sensitive ECL substrates for optimal detection of phosphorylated tau

• Controls: Include both positive controls (samples known to contain phosphorylated tau at S519) and negative controls (dephosphorylated samples)

How can I use Phospho-MAPT (S519) Antibody to distinguish between different tauopathies?

Different tauopathies exhibit distinct patterns of tau phosphorylation:

• Comparative phosphorylation profiling: Use a panel of phospho-tau antibodies targeting different sites alongside the S519 antibody to create phosphorylation signatures specific to different tauopathies .

• Co-localization studies: Combine Phospho-MAPT (S519) Antibody with antibodies against other disease markers to establish disease-specific patterns.

• Temporal analysis: Monitor changes in S519 phosphorylation at different disease stages to determine if it's an early or late event in pathogenesis.

• Regional distribution: Map the brain regions where S519 phosphorylated tau appears in different conditions like Alzheimer's disease versus Progressive Supranuclear Palsy (PSP) .

The MDS-PSP criteria for diagnosing Progressive Supranuclear Palsy notes: "The abundance of brain tau aggregates correlates with disease severity and select phospho-tau epitopes increase at early stages of disease" . Determining if S519 is among these early markers can provide valuable diagnostic insights.

What considerations are important when using Phospho-MAPT (S519) Antibody in animal models of tauopathy?

When using this antibody in animal models:

• Species cross-reactivity: Verify the antibody recognizes the target in your model species. Most Phospho-MAPT (S519) antibodies react with human, mouse, and rat tau , but sequence conservation should be confirmed.

• Model selection: Different models express different tau isoforms and show varied phosphorylation patterns:

  • Human tau (hTau) transgenic mice express all six human tau isoforms

  • Some models express both mouse and human tau

  • Tau knockout mice with human tau knock-in provide cleaner backgrounds

• Timing considerations: Consider the time lag between mRNA and protein changes. Research shows "tau protein levels lagged behind the changes in transcript, with peak lowering of 55% detected at 8 weeks post dose... This observation fits with a longer in vivo half-life for the tau protein" .

• Regional analysis: Different brain regions show varied tau expression and phosphorylation. "Broad distribution of ASO-001933 was detected by in situ hybridization histochemistry (ISH) using a fluorescently labeled sense probe to ASO-001933, with the highest signal in cortical regions" .

How can I optimize immunohistochemistry protocols for detecting phosphorylated tau at S519 in tissue sections?

For optimal immunohistochemical detection:

• Fixation: Use 10% neutral buffered formalin fixation for 24-48 hours, followed by standard paraffin embedding

• Antigen retrieval: Critical for phospho-epitopes; use citrate buffer (pH 6.0) and heat-induced epitope retrieval

• Blocking endogenous peroxidase: Treat sections with 3% hydrogen peroxide before antibody incubation

• Background reduction: Block with 5% normal serum from the same species as the secondary antibody

• Primary antibody optimization:

  • Start with 1:500 dilution and optimize as needed

  • Incubate overnight at 4°C for maximum sensitivity

• Signal amplification: Consider using tyramide signal amplification for low abundance phospho-epitopes

• Counterstaining: Use a light hematoxylin counterstain to avoid obscuring DAB signal

As noted in screening methods for novel monoclonal antibodies: "The positive clones were next screened by immunohistochemistry of a human AD autopsy case with abundant tau pathology" , indicating the value of this application in tauopathy research.

What are common pitfalls when working with phospho-specific antibodies like Phospho-MAPT (S519)?

Common challenges and solutions include:

• Loss of phosphorylation during sample preparation:

  • Always include phosphatase inhibitors in lysis buffers

  • Maintain samples at 4°C during processing

  • Avoid multiple freeze-thaw cycles of samples

• Cross-reactivity with other phosphorylation sites:

  • Validate specificity with phosphopeptide competition assays

  • Use site-directed mutants as negative controls

• Antibody specificity drift over time:

  • Aliquot antibodies upon receipt to minimize freeze-thaw cycles

  • Re-validate antibodies with positive controls periodically

  • Include appropriate controls in each experiment

• Balancing phospho-recognition and sequence recognition:
  "A balance in stability of phospho-recognition and sequence recognition is critical for specificity. A single CDR that captures the phosphate group imparts the ability to weakly interact with a wide range of phospho-peptides to antibodies" .

How can I design experiments to study the relationship between tau phosphorylation at S519 and other phosphorylation sites?

To investigate interrelationships between tau phosphorylation sites:

• Sequential immunoprecipitation:

  • First immunoprecipitate with Phospho-MAPT (S519) Antibody

  • Then probe the immunoprecipitate with antibodies against other phospho-sites

  • This reveals co-occurrence of multiple phosphorylation events on the same tau molecules

• Kinase/phosphatase manipulation:

  • Treat samples with specific kinase activators or inhibitors

  • Monitor how manipulation of one pathway affects phosphorylation at S519 and other sites

  • This helps establish hierarchical relationships between phosphorylation events

• Time-course experiments:

  • Induce tau phosphorylation and monitor the temporal sequence of phosphorylation at different sites

  • This establishes which sites are phosphorylated early versus late in the pathological process

• Site-directed mutagenesis:

  • Generate tau constructs with mutations at specific phosphorylation sites

  • Observe how preventing phosphorylation at one site affects other sites

  • This reveals interdependencies between phosphorylation events

What considerations are important when designing therapeutic interventions targeting tau phosphorylation?

When designing therapeutics targeting phosphorylated tau:

• Temporal and spatial expression: Understand when and where S519 phosphorylation occurs in disease progression

• Functional consequences: Determine whether S519 phosphorylation is a driver or consequence of pathology

• Targeting approaches: Consider strategies like antisense oligonucleotides (ASOs) which have shown promise in tau reduction

• Treatment monitoring: Use Phospho-MAPT (S519) Antibody to monitor therapeutic efficacy in reducing target phosphorylation

Research with tau-targeting ASOs demonstrates important principles: "ASO-001933 potently reduced tau mRNA and protein in mice... A single dose of 100 μg of ICV resulted in a 56% tau mRNA reduction 1 week post dose... Protein lowering was not yet detected at this time point, similar to what was observed in C57Bl/6J mice" . This highlights the importance of understanding the relationship between intervention, gene expression, and protein levels when designing therapeutic approaches.

How might Phospho-MAPT (S519) Antibody be used in developing novel biomarkers for neurodegenerative diseases?

Emerging applications include:

• Development of ultrasensitive assays: New high-sensitivity detection methods could enable detection of minute amounts of phosphorylated tau at S519 in cerebrospinal fluid or blood .

• Digital biomarker platforms: Integration with digital biomarker platforms for comprehensive patient profiling and disease monitoring.

• Combination biomarker panels: Incorporation of S519 phosphorylation status with other tau phosphorylation sites and additional biomarkers for improved diagnostic accuracy.

• PET tracer development: Insights from antibody binding properties could inform development of positron emission tomography (PET) tracers specific for phosphorylated tau.

Recent research notes: "An essential component of high-sensitivity immunoassays is antibodies that selectively recognize the target in complex samples... Achieving p-tau specificity is particularly challenging since the antibodies need to distinguish the presence of a single phosphorylated residue" .

What new methodologies are being developed to improve the specificity and sensitivity of phospho-tau detection?

Cutting-edge approaches include:

• Antibody engineering: "Based on the fact that not all variants with improved affinity showed binding to the non-phospho-peptide, a second stage of screening was conducted for the absence of non-phospho-peptide binding. This led to the identification of a high-specificity pThr231 tau antibody with a picomolar dissociation constant" . Similar approaches could be applied to S519 antibodies.

• Novel signal amplification methods: Enhanced chemiluminescence, tyramide signal amplification, and quantum dot labeling for dramatically improved sensitivity.

• Single-molecule detection platforms: Application of technologies like Simoa (single molecule array) for ultrasensitive detection of phosphorylated tau in biological fluids.

• Proximity-based assays: Development of proximity extension or proximity ligation assays that require dual antibody binding for signal generation, enhancing specificity.

• Structural insights: "The main feature of these antibodies is their tight association with the phosphate group of the modified residues... antibodies engage the phosphate group using diverse complementarity determining region (CDR) residues including those in CDR H1, H2, H3, or L1" . These structural insights are driving rational design of next-generation antibodies.