Phospho-MAPT (S516/199) Antibody

Basic Research Methodology Questions

• What is the specificity profile of Phospho-MAPT (S516/199) Antibody and how should it be validated?

The Phospho-MAPT (S516/199) Antibody specifically recognizes tau protein phosphorylated at serine residues 516 and 199, without cross-reactivity to other proteins as indicated in the product specifications . To properly validate this antibody's specificity, researchers should:

• Perform Western blot analysis using both phosphorylated and non-phosphorylated tau protein samples

• Include positive controls using human, mouse, or rat brain lysates with known tau pathology

• Run parallel validation with established phospho-tau antibodies (such as PHF-1 or AT100)

• Use a peptide competition assay with the synthesized immunogen peptide to confirm specificity

• Verify reactivity using ELISA with peptides containing the phosphorylated residues versus non-phosphorylated controls

These validation steps are essential to ensure the antibody recognizes the intended phosphorylation sites without cross-reactivity to other phosphorylation sites on tau or other proteins.

• What are the optimal experimental conditions for using Phospho-MAPT (S516/199) Antibody in Western blotting?

For optimal Western blot performance with Phospho-MAPT (S516/199) Antibody, researchers should follow these methodological guidelines:

• Recommended dilution range: 1:500-1:2000 for Western blotting

• Buffer composition: Use PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide for antibody dilution

• Sample preparation: For brain tissues, use sarkosyl-insoluble fractions to enrich for pathological tau

• Blocking solution: 5% non-fat dry milk or BSA in TBST is typically effective

• Detection system: HRP-conjugated secondary antibodies with enhanced chemiluminescence provide suitable sensitivity

• Loading controls: Include total tau antibody on parallel blots to normalize phospho-tau signals

• Positive controls: Include samples from AD brain tissues or tau transgenic mouse models

Researchers should note that the calculated molecular weight of tau is approximately 78928 Da, but due to post-translational modifications and isoform variations, the observed molecular weight may vary considerably on Western blots .

• What applications beyond Western blotting are validated for Phospho-MAPT (S516/199) Antibody?

The Phospho-MAPT (S516/199) Antibody has been validated for multiple experimental techniques beyond Western blotting:

• ELISA: Recommended at a dilution of 1:40000 for high sensitivity detection

• Immunohistochemistry: While not explicitly mentioned for this specific antibody in the provided resources, similar phospho-tau antibodies are routinely used in IHC applications to visualize tau inclusions in brain tissue sections

• Immunocytochemistry: Can be applied to cultured neurons or cell models expressing tau proteins

• Immunoprecipitation: Useful for isolating phosphorylated tau proteins for further analysis

Each application requires specific optimization, particularly regarding fixation methods, antigen retrieval techniques, and antibody concentrations. For novel applications, researchers should perform preliminary titration experiments to determine optimal conditions .

Advanced Research Methodology Questions

• How can Phospho-MAPT (S516/199) Antibody be applied in tau seeding assays to investigate tauopathy progression mechanisms?

Phospho-MAPT (S516/199) Antibody can be strategically implemented in tau seeding assays to investigate pathological tau propagation mechanisms:

• Seed preparation methodology:

  • Use sarkosyl-insoluble fractions from tauopathy brain samples or aged transgenic mouse models as seeds

  • Perform biochemical fractionation through sucrose gradient ultracentrifugation to isolate different tau aggregate species

  • Quantify phosphorylated tau at S516/199 by ELISA in the seed preparations to normalize seeding activity

• Seeding detection approaches:

  • Monitor S516/199 phosphorylation as an early marker of successful seeding using the antibody

  • Perform time-course experiments to track the progression of S516/199 phosphorylation after seeding

  • Compare with other phosphorylation sites to establish temporal phosphorylation patterns

• Experimental design considerations:

  • In cellular models, transfect cells with wild-type or mutant tau (particularly focusing on P301 mutations which show enhanced seeding properties)

  • For in vivo models, use stereotactic injection into hippocampus or cortex, as shown effective in multiple models

  • Consider the impact of tau isoform differences, as 3R vs. 4R tau show different seeding barriers

This table summarizes key experimental parameters for tau seeding assays based on research findings:

• What are the methodological differences in detecting S516/199 phosphorylation compared to other phospho-tau epitopes in neurodegenerative disease research?

Methodological approaches for detecting S516/199 phosphorylation differ from other phospho-tau epitopes in several important aspects:

• Sample preparation considerations:

  • For optimal S516/199 detection, samples should be rapidly preserved to prevent post-mortem dephosphorylation

  • Phosphatase inhibitors (including sodium fluoride, sodium pyrophosphate, and β-glycerophosphate) must be included in all extraction buffers

  • Heat-stable fractionation methods may be more suitable for certain phospho-epitopes but not others

• Epitope-specific biochemical properties:

  • S516/199 phosphorylation appears in specific tau aggregation stages that may differ from other sites

  • Unlike the AT8 epitope (pSer202/pThr205) which appears early in pretangle neurons, the temporal appearance of S516/199 phosphorylation in disease progression requires further characterization

  • Certain phospho-epitopes show differential solubility in sarkosyl extraction, affecting detection methods

• Comparative analysis with established phospho-tau markers:

  • The PHF-1 antibody (pSer396/pSer404) has been extensively characterized for detecting late-stage tau pathology

  • The AT100 antibody, which recognizes tau phosphorylated at both Thr212 and Ser214, serves as a positive control in many studies

  • Novel conformation-dependent tau antibodies like 5E2 and 2F12 display similar specificity to PHF-1 for various tauopathies

• Technical validation approaches:

  • In vitro kinase assays using GSK3β or MAPK can generate specifically phosphorylated tau for antibody validation

  • Dephosphorylation treatments with alkaline phosphatase can confirm phospho-specificity of antibody binding

  • Peptide competition assays with phosphorylated versus non-phosphorylated peptides provide definitive specificity validation

• How do MAPT mutations influence the detection of S516/199 phosphorylation and what methodological adaptations are required?

MAPT mutations significantly impact tau phosphorylation patterns and detection methodologies, requiring specific experimental adaptations when using Phospho-MAPT (S516/199) Antibody:

• Effects of specific MAPT mutations on S516/199 phosphorylation:

  • P301L/S/T mutations enhance tau aggregation propensity and may alter phosphorylation kinetics at multiple sites including S516/199

  • The S320F mutation notably increases tau's aggregation tendency even without seeding, which may affect phosphorylation patterns

  • Researchers must consider how mutations alter the conformational landscape of tau and potentially mask or expose the S516/199 epitope

• Methodology adaptations for mutant tau detection:

  • When studying P301 mutants, lower antibody concentrations may be required due to enhanced phosphorylation signals

  • For S320F mutants, shorter incubation periods with phospho-specific antibodies may be sufficient

  • Double mutant combinations (e.g., P301L/S with S320F) may require further protocol adjustments due to rapid and robust aggregation even without seeding

• Comparative analysis approaches:

  • Always run parallel samples of wild-type and mutant tau to accurately assess differences in phosphorylation profiles

  • Use multiple phospho-tau antibodies targeting different epitopes to create a comprehensive phosphorylation map

  • Consider the use of site-specific mutagenesis (S→A) controls to verify antibody specificity in mutant backgrounds

• Specialized techniques for complex mutation scenarios:

  • For mutations that potentially alter epitope accessibility, mild denaturation steps may improve detection

  • Native PAGE versus SDS-PAGE can reveal how mutations affect conformational states that influence epitope accessibility

  • Mass spectrometry validation of phosphorylation sites provides definitive confirmation in complex mutant scenarios

Research has demonstrated that proline residues can serve as inhibitors of β-sheet formation, with the P301 position having a uniquely important role in preventing pathological tau aggregation . When investigating mutants at this position, researchers should carefully interpret phosphorylation data in the context of the enhanced aggregation propensity these mutations confer.

• What are the optimal protocols for applying Phospho-MAPT (S516/199) Antibody in studies of tau pathology progression?

Optimal protocols for applying Phospho-MAPT (S516/199) Antibody in tau pathology progression studies require careful consideration of multiple methodological factors:

• Tissue preparation approaches:

  • For human brain tissue: 10% neutral buffered formalin fixation for 24-48 hours followed by paraffin embedding

  • For rodent models: Transcardial perfusion with 4% paraformaldehyde, followed by post-fixation

  • Antigen retrieval optimization: Test multiple methods including citrate buffer (pH 6.0), EDTA buffer (pH 8.0), and formic acid treatment

  • Section thickness: 5-7 μm sections for standard IHC; 40-60 μm for free-floating sections

• Immunodetection methodology:

  • Primary antibody incubation: 1:500-1:2000 dilution at 4°C overnight for optimal signal-to-noise ratio

  • Secondary detection systems: Biotinylated secondary antibody followed by ABC complex or polymer-based detection systems

  • DAB development: Carefully timed exposure (3-5 minutes) for consistent staining intensity

  • Counterstaining: Light hematoxylin counterstain for cellular context

• Comparative analysis framework:

  • Multiple brain regions: Analyze hippocampus, entorhinal cortex, and neocortical regions to track pathology spread

  • Sequential sectioning: Stain adjacent sections with multiple phospho-tau markers (AT8, PHF-1, AT100) to compare epitope appearance

  • Quantification methods: Digital image analysis with threshold-based quantification of immunoreactive area

• Longitudinal study design considerations:

  • Time points selection: For mouse models, analyze at 2, 4, 6, and 12 months of age

  • Systematic sampling: Use unbiased stereological approaches for quantitative analysis

  • Correlation with behavioral deficits: Align tissue analysis timepoints with behavioral assessment

When implementing these protocols, researchers should note that PHF-1 and similar antibodies have shown specificity for Alzheimer's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and control cases for specific brain regions investigated , suggesting that careful regional analysis is critical when applying Phospho-MAPT (S516/199) Antibody to distinguish between different tauopathies.

• How can researchers effectively troubleshoot non-specific binding issues with Phospho-MAPT (S516/199) Antibody?

When troubleshooting non-specific binding issues with Phospho-MAPT (S516/199) Antibody, researchers should implement a systematic methodology:

• Antibody validation and quality control:

  • Verify antibody lot performance using positive control samples (AD brain tissue)

  • Perform peptide competition assays with both phosphorylated and non-phosphorylated peptides

  • Use phosphatase treatment controls to confirm phospho-specificity

• Sample preparation optimization:

  • Ensure complete phosphatase inhibition during tissue/cell lysis (cocktail including NaF, Na3VO4, and β-glycerophosphate)

  • Optimize protein extraction methods based on tau solubility (RIPA buffer vs. sarkosyl extraction)

  • For fixed tissues, test multiple antigen retrieval protocols systematically

• Protocol adjustment strategies:

  • Blocking optimization: Test 5% BSA vs. 5% normal serum from secondary antibody host species

  • Antibody dilution series: Perform serial dilutions from 1:250 to 1:5000 to identify optimal concentration

  • Buffer optimization: Add 0.1% Tween-20 to PBS for reduced background

  • Incubation conditions: Compare room temperature (1-2 hours) vs. 4°C overnight incubation

• Advanced troubleshooting techniques:

  • Pre-adsorption: Incubate antibody with non-relevant tissue lysates to remove non-specific binding components

  • Secondary antibody controls: Perform control staining without primary antibody

  • Cross-adsorbed secondary antibodies: Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity

  • Sequential staining approach: For co-localization studies, use sequential rather than simultaneous antibody incubations

The product specifications indicate that Phospho-MAPT (S516/199) Antibody shows no cross-reactivity with other proteins , but researchers should remain vigilant about potential non-specific binding in complex biological samples, particularly in tissues with high lipid content such as brain.

Research Applications and Technical Questions

• What are the comparative advantages of using Phospho-MAPT (S516/199) Antibody versus other phospho-tau antibodies in tauopathy research?

The comparative advantages of Phospho-MAPT (S516/199) Antibody versus other established phospho-tau antibodies must be considered within specific research contexts:

• Epitope-specific advantages:

  • Unlike AT8 (pSer202/pThr205) which detects early pre-tangle changes, S516/199 phosphorylation may represent distinct stages in tau pathology

  • Compared to PHF-1 (pSer396/pSer404), which is widely used for late-stage tangles, S516/199 provides complementary information about tau's phosphorylation state

  • The dual-epitope nature (recognizing both S516 and S199) may provide unique insights into the coordination of phosphorylation events in disease progression

• Technical performance comparisons:

  • Validated for both Western blot (1:500-1:2000) and ELISA (1:40000) applications, offering versatility across multiple techniques

  • Documented reactivity across multiple species (human, mouse, rat), facilitating translational research between animal models and human samples

  • The polyclonal nature potentially recognizes multiple conformational states of the phosphorylated epitopes

• Research application advantages:

  • Valuable for studying the effects of tau mutations, particularly at P301 sites which show enhanced seeding properties

  • Useful in tau seeding experiments to track pathology spread, as demonstrated in multiple mouse models

  • Applicable to diverse tauopathies beyond Alzheimer's disease, including corticobasal degeneration and progressive supranuclear palsy

• Methodological advantages:

  • The immunogen design (synthesized peptide derived from human Tau around the phosphorylation site of S516/199) provides high specificity

  • Compatible with standard immunohistochemistry protocols used for other phospho-tau antibodies

  • Usable in conjunction with tau conformation-specific antibodies for multi-dimensional characterization of tau pathology

Research has shown that phosphorylation of tau at specific residues, such as those in the AT8 and PHF1 epitopes, occurs early in tau inclusion formation , making the comparative analysis of multiple phospho-epitopes, including S516/199, particularly valuable for understanding the temporal progression of tauopathies.

• How does the phosphorylation of tau at S516/199 sites correlate with tau aggregation mechanisms in different tauopathies?

The correlation between S516/199 phosphorylation and tau aggregation mechanisms in different tauopathies involves complex molecular interactions:

• Mechanistic relationship with tau aggregation:

  • Phosphorylation at specific sites like S516/199 may disrupt tau's protective paperclip-like global conformation, potentially facilitating polymerization

  • Similar to other phosphorylation sites, S516/199 phosphorylation likely reduces tau's binding affinity for microtubules, increasing the pool of free tau available for aggregation

  • The specific contribution of S516/199 phosphorylation to nucleation versus elongation phases of aggregation requires further characterization

• Tauopathy-specific phosphorylation patterns:

  • In Alzheimer's disease (AD), cryo-electron microscopy has revealed that specific residues including S320 reside within hydrophobic pockets of tau filaments

  • Pick's disease (PiD) tau filaments show distinct structural arrangements compared to AD, potentially affecting the accessibility and importance of S516/199 phosphorylation

  • Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) involve predominantly 4R tau isoforms, which may influence the significance of specific phosphorylation sites

• Connection to tau strains and conformation:

  • Different "strains" of tau aggregates can survive multiple passages through cell culture and mice , suggesting stable conformational templating that may involve specific phosphorylation patterns

  • Tau seeding experiments have shown that isoform differences (3R versus 4R tau) create seeding barriers , which may be influenced by phosphorylation status

  • Phosphorylation at S516/199 may contribute to strain-specific conformations of tau aggregates

• Temporal dynamics in disease progression:

  • The disruption of tau's paperclip-like structure has been demonstrated in vitro with pseudo-phosphorylation at the AT8 and PHF1 epitopes

  • S516/199 phosphorylation may occur at specific stages in the cascade of post-translational modifications leading to mature tau aggregates

  • The sequence of phosphorylation events (rather than individual sites) may be critical for pathological aggregation

Research on mutant tau has shown that specific mutations (such as P301L/S and S320F) significantly affect aggregation properties , suggesting that the relationship between phosphorylation and aggregation is influenced by the primary sequence context and may vary across different tauopathies.