Phospho-MAPT (S214) Recombinant Monoclonal Antibody

What is the significance of MAPT (Tau) phosphorylation at Ser214 in neurodegenerative research?

Phosphorylation of MAPT (microtubule-associated protein tau) at Ser214 (pTAU-S214) represents a critical post-translational modification with significant implications for neurodegenerative diseases. Unlike phosphorylation at other sites that promote pathological tau assembly, phosphorylation at Ser214 has been demonstrated to reduce the pathological assembly of the protein . This site-specific phosphorylation event is primarily mediated by cAMP-dependent protein kinase A (PKA) . Understanding pTAU-S214 is particularly relevant for Alzheimer's disease and other tauopathies, as it potentially represents a protective modification that could be therapeutically enhanced to reduce tau aggregation and its associated synapto/neurotoxic effects .

What are the key characteristics of Phospho-MAPT (S214) antibodies used in research?

Phospho-MAPT (S214) antibodies are specialized immunological tools designed to detect MAPT specifically when it is phosphorylated at the Ser214 residue. These antibodies typically demonstrate the following characteristics:

The choice between polyclonal and recombinant monoclonal antibodies should be based on experimental requirements, with monoclonals generally offering higher specificity and reproducibility for quantitative analyses .

How should I design experiments to study cAMP-dependent phosphorylation of MAPT at Ser214?

When investigating cAMP-dependent phosphorylation of MAPT at Ser214, a systematic experimental approach is required. Based on validated methodologies, consider the following experimental design:

• Define your variables carefully:

  • Independent variable: Concentration of cAMP enhancers (e.g., forskolin, GEBR-7b)

  • Dependent variable: Level of tau phosphorylation at Ser214

  • Control variables: Cell type, treatment duration, temperature, culture conditions

• Establish appropriate model systems:

  • Cell culture models (e.g., N2a neuronal cells) for initial screening

  • Ex vivo tissue preparations (e.g., hippocampal slices) to validate findings in more complex systems

• Implement experimental treatments:

  • Use adenylyl cyclase activators (like forskolin) to increase cAMP synthesis

  • Apply phosphodiesterase inhibitors (such as GEBR-7b, a PDE4D inhibitor) to prevent cAMP degradation

  • Consider combination treatments to maximize effects

• Include appropriate controls:

  • Vehicle controls for all treatments

  • Positive controls (treatments known to induce pTAU-S214)

  • Negative controls (treatments that block PKA activity)

• Perform quantitative analysis:

  • Western blotting with Phospho-MAPT (S214) antibodies

  • Normalize phospho-tau levels to total tau protein

  • Apply appropriate statistical tests to determine significance of differences between treatment groups

This approach allows for systematic investigation of cAMP's role in tau phosphorylation at Ser214 while controlling for potential confounding variables .

What considerations are important when validating Phospho-MAPT (S214) antibody specificity?

Validating antibody specificity is crucial for obtaining reliable and reproducible results in tau phosphorylation research. For Phospho-MAPT (S214) antibodies, consider implementing these validation strategies:

• Phosphatase treatment controls:

  • Treat sample aliquots with lambda phosphatase to remove phosphate groups

  • Compare antibody reactivity between phosphatase-treated and untreated samples

  • Expect significant reduction in signal after phosphatase treatment

• Peptide competition assays:

  • Pre-incubate antibody with excess phosphorylated and non-phosphorylated peptides containing the S214 epitope

  • Observe whether phospho-peptide specifically blocks antibody binding

  • Non-phosphorylated peptide should not affect antibody binding

• Phosphomimetic mutants:

  • Test antibody against S214A (non-phosphorylatable) and S214E/D (phosphomimetic) tau mutants

  • Confirm antibody recognizes phosphomimetic but not alanine mutant

• Kinase activation/inhibition:

  • Stimulate PKA with cAMP enhancers (e.g., forskolin) to increase S214 phosphorylation

  • Inhibit PKA to decrease phosphorylation

  • Verify antibody signal increases with PKA activation and decreases with inhibition

• Cross-reactivity assessment:

  • Test antibody against samples containing other phosphorylated proteins

  • Ensure no recognition of similar phosphorylation motifs in unrelated proteins

These validation steps ensure that experimental outcomes truly reflect specific detection of pTAU-S214 rather than non-specific binding or artifacts .

How can Phospho-MAPT (S214) antibodies be used to investigate the relationship between cAMP signaling and tau pathology?

Phospho-MAPT (S214) antibodies serve as powerful tools for exploring the mechanistic links between cAMP signaling and tau pathology in neurodegenerative diseases. A comprehensive research approach might include:

• Signaling pathway dissection:

  • Use Phospho-MAPT (S214) antibodies to quantify phosphorylation levels following manipulation of various components of the cAMP/PKA pathway

  • Apply specific activators (forskolin) and inhibitors (GEBR-7b) of adenylyl cyclase and phosphodiesterases to modulate cAMP levels

  • Determine dose-response relationships and temporal dynamics of S214 phosphorylation following cAMP elevation

• Neuroprotection studies:

  • Induce tau aggregation in cellular or animal models of tauopathy

  • Treat with cAMP enhancers to increase S214 phosphorylation

  • Use Phospho-MAPT (S214) antibodies to correlate phosphorylation levels with reduction in tau aggregation and neurotoxicity

  • Assess whether S214 phosphorylation correlates with improved neuronal survival and function

• Cross-talk analysis:

  • Investigate interactions between cAMP signaling and other pathways that regulate tau phosphorylation

  • Examine how S214 phosphorylation affects other tau phosphorylation sites (e.g., S202)

  • Use Phospho-MAPT (S214) antibodies in conjunction with antibodies against other phospho-epitopes to build a comprehensive phosphorylation profile

• Amyloid-β independence:

  • Employ γ-secretase inhibitors (like compound E) to block Aβ production

  • Confirm that cAMP-induced S214 phosphorylation occurs independently of Aβ peptides

  • Use Phospho-MAPT (S214) antibodies to quantify phosphorylation levels under these conditions

This multifaceted approach leverages Phospho-MAPT (S214) antibodies to elucidate the complex relationship between cAMP signaling and tau pathophysiology, potentially identifying novel therapeutic targets for neurodegenerative diseases .

What methodological approaches can resolve contradictory findings when studying Ser214 phosphorylation?

Contradictory findings in tau phosphorylation research can stem from methodological differences, biological variability, or context-dependent effects. To resolve such discrepancies when studying Ser214 phosphorylation, consider these systematic approaches:

• Standardize experimental conditions:

  • Control for cellular activation state, as basal cAMP levels significantly impact the effects of phosphodiesterase inhibitors

  • Note that in N2a cells, GEBR-7b alone may not significantly increase pTAU-S214 due to low basal cAMP levels, while in hippocampal slices with more active synaptic circuits, GEBR-7b can increase pTAU-S214 without forskolin stimulation

  • Standardize cell density, passage number, and culture conditions

• Employ multiple model systems:

  • Compare findings across different experimental models (cell lines, primary neurons, tissue slices, in vivo models)

  • Recognize that pathway regulation may differ between simplified in vitro systems and complex tissues

  • For example, PKA may phosphorylate tau directly in some contexts but activate intermediate kinases in others

• Conduct time-course analyses:

  • Implement temporal profiling of phosphorylation events

  • Determine whether contradictory findings reflect different time points in dynamic phosphorylation processes

  • Monitor both rapid (minutes to hours) and prolonged (days) responses

• Consider context-dependent effects:

  • Investigate whether disease state, age, or stress conditions alter the relationship between cAMP signaling and S214 phosphorylation

  • Examine whether priming phosphorylation at other sites influences S214 phosphorylation

• Apply quantitative biochemical techniques:

  • Use multiple detection methods beyond Western blotting (mass spectrometry, ELISA, immunohistochemistry)

  • Implement absolute quantification of phosphorylation stoichiometry

  • Normalize phosphorylation levels appropriately (to total tau rather than housekeeping proteins)

By implementing these methodological approaches, researchers can better understand context-dependent effects and resolve apparent contradictions in the study of Ser214 phosphorylation of tau .

What are the optimal conditions for using Phospho-MAPT (S214) antibodies in various applications?

Achieving optimal results with Phospho-MAPT (S214) antibodies requires application-specific optimization. The following guidelines are based on validated protocols:

Western Blotting (WB)

• Sample preparation: Preserve phosphorylation status by including phosphatase inhibitors in lysis buffers

• Blocking: Use 5% BSA in TBST (not milk, which contains phosphatases)

• Antibody dilution: Start with 1:1000 dilution (1 μg/ml) and optimize as needed

• Incubation: Overnight at 4°C for primary antibody

• Detection: HRP-conjugated secondary antibodies with enhanced chemiluminescence

Enzyme-Linked Immunosorbent Assay (ELISA)

• Coating concentration: 1-2 μg/ml of capture antibody

• Sample dilution: Prepare a dilution series to determine optimal concentration

• Blocking: 1-3% BSA in PBS or TBS

• Detection: Use HRP-conjugated secondary antibody or biotinylated detection antibody with streptavidin-HRP

Immunohistochemistry (IHC)

• Fixation: 4% paraformaldehyde is recommended

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Blocking: 5-10% normal serum from the same species as the secondary antibody

• Antibody dilution: Begin with 1:100-1:500 dilution

• Incubation: Overnight at 4°C for primary antibody

Storage and Handling

• Store antibody at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• Prepare working aliquots for frequent use

• Working dilutions can typically be stored at 4°C for up to one month

These conditions provide starting points for optimization, which should be tailored to specific experimental requirements and sample types.

How can I determine whether changes in Ser214 phosphorylation are causally related to tau aggregation or disease progression?

Establishing causal relationships between Ser214 phosphorylation and tau aggregation requires sophisticated experimental approaches that go beyond correlation. Consider implementing these methodological strategies:

• Site-directed mutagenesis studies:

  • Generate phosphomimetic (S214E/D) and phospho-deficient (S214A) tau mutants

  • Express these in cellular or animal models of tauopathy

  • Compare aggregation propensity, microtubule binding, and neurotoxicity between variants

  • Use Phospho-MAPT (S214) antibodies to confirm absence of phosphorylation in S214A mutants

• Temporal sequence analysis:

  • Conduct time-course experiments in disease models

  • Map the sequence of phosphorylation events, aggregation, and cellular dysfunction

  • Determine whether S214 phosphorylation precedes or follows other disease markers

  • Use Phospho-MAPT (S214) antibodies to track phosphorylation status throughout disease progression

• Pharmacological intervention studies:

  • Modulate cAMP/PKA signaling to alter S214 phosphorylation

  • Assess downstream effects on tau aggregation and neurodegeneration

  • Implement rescue experiments to determine whether enhancing S214 phosphorylation can reverse established pathology

  • Apply Phospho-MAPT (S214) antibodies to confirm target engagement

• Structure-function analyses:

  • Investigate how S214 phosphorylation alters tau conformation

  • Perform in vitro aggregation assays with recombinant tau proteins

  • Conduct molecular dynamic simulations to understand conformational changes

  • Consider that priming phosphorylation of Ser214 by PKA protects other sites of tau from phosphorylation by glycogen synthase kinase 3β, potentially preventing pathological PHF-like conformations

• In vivo genetic manipulation:

  • Develop knock-in mouse models expressing S214 variants

  • Assess effects on age-dependent tau pathology and cognitive function

  • Use conditional expression systems to control timing of mutation expression

These methodologies provide complementary approaches to establish whether S214 phosphorylation plays a causative role in preventing tau aggregation and disease progression, or merely represents a correlative marker .

How might the relationship between cAMP signaling and tau phosphorylation at Ser214 inform therapeutic strategies for tauopathies?

The relationship between cAMP signaling and Ser214 phosphorylation offers promising avenues for therapeutic intervention in tauopathies. Current research suggests several strategic approaches:

• Targeted phosphodiesterase inhibition:

  • PDE4D inhibitors like GEBR-7b enhance cAMP signaling and increase Ser214 phosphorylation

  • These compounds have demonstrated pro-cognitive efficacy in rodent models

  • Selective PDE inhibition may provide more targeted effects with fewer side effects than global cAMP elevation

  • The combined use of adenylyl cyclase activators with PDE inhibitors may produce synergistic effects on S214 phosphorylation

• PKA activation strategies:

  • Direct or indirect activation of PKA could increase S214 phosphorylation

  • Development of PKA activators with improved blood-brain barrier penetration

  • Investigation of natural compounds that enhance PKA activity in neurons

• Protection against pathological phosphorylation:

  • Priming phosphorylation at Ser214 by PKA protects other tau sites from phosphorylation by GSK3β

  • This mechanism prevents the PHF-like conformation of tau

  • Therapeutic approaches could focus on enhancing this protective priming effect

  • Combined modulation of PKA (to increase S214 phosphorylation) and GSK3β (to decrease pathological phosphorylation)

• Integration with amyloid-targeted therapies:

  • Since cAMP-induced S214 phosphorylation occurs independently of Aβ peptides

  • These approaches may complement anti-amyloid strategies

  • Combination therapies targeting both pathways might provide synergistic benefits

  • Patient stratification based on tau versus amyloid pathology prominence could guide personalized treatment

• Biomarker development:

  • Phospho-MAPT (S214) antibodies could be developed for diagnostic applications

  • Monitoring S214 phosphorylation status as a response biomarker for cAMP-enhancing therapies

  • Potential for PET imaging ligands targeting pTAU-S214 to visualize therapeutic effects in vivo

These therapeutic strategies highlight the potential of targeting cAMP-mediated S214 phosphorylation as a novel approach to reducing tau aggregation and associated neurotoxicity in tauopathies .

What are the methodological considerations for studying the interplay between different tau phosphorylation sites, including Ser214?

Investigating the complex interplay between multiple tau phosphorylation sites requires sophisticated methodological approaches. Researchers should consider these strategies when studying how Ser214 phosphorylation interacts with other modifications:

• Multiplexed phosphorylation analysis:

  • Employ antibody panels targeting multiple phospho-epitopes simultaneously

  • Utilize mass spectrometry-based phosphoproteomics to quantify multiple phosphorylation sites

  • Apply phospho-specific flow cytometry for single-cell resolution of multiple phosphorylation events

  • Design experiments to detect sequential phosphorylation patterns and site interdependencies

• Kinase/phosphatase manipulation:

  • Selectively activate or inhibit specific kinases (PKA, GSK3β, CDK5, MARK)

  • Map the resulting changes across the tau phosphorylation landscape

  • Determine whether Ser214 phosphorylation by PKA affects subsequent phosphorylation events by other kinases

  • Consider that priming phosphorylation of Ser214 by PKA protects other sites (Thr212 and ser-pro motifs around residue 200) from GSK3β phosphorylation

• Structural biology approaches:

  • Implement NMR studies to determine how phosphorylation at one site affects protein conformation

  • Use FRET-based biosensors to detect conformational changes in living cells

  • Apply cryo-EM to visualize different phosphorylated states of tau filaments

  • Develop computational models to predict how combinations of phosphorylation events affect protein structure

• Temporal dynamics investigation:

  • Design pulse-chase experiments to track sequential phosphorylation events

  • Implement rapid kinetic analyses to determine order of phosphorylation

  • Develop biosensors for real-time monitoring of phosphorylation in living cells

  • Consider both rapid signaling events and long-term accumulation of phosphorylated species

• Multi-omics integration:

  • Combine phosphoproteomics with transcriptomics and metabolomics

  • Identify signaling networks and feedback mechanisms controlling tau phosphorylation

  • Map how changes in one phosphorylation site propagate through cellular signaling networks

  • Correlate phosphorylation patterns with cellular phenotypes and disease progression

These methodological approaches provide complementary strategies for unraveling the complex interplay between Ser214 and other phosphorylation sites, potentially revealing novel intervention points for tauopathies .