Phospho-MAPT (Ser404) Antibody

What is Phospho-MAPT (Ser404) Antibody and what epitope does it recognize?

Phospho-MAPT (Ser404) Antibody specifically detects endogenous levels of tau protein only when phosphorylated at serine 404. The antibody recognizes the peptide sequence around the phosphorylation site of serine 404 (D-T-S(p)-P-R) derived from human tau . It's important to note that this antibody does not recognize non-phosphorylated tau at this position, making it highly specific for the phosphorylated form.

The antibody targets the C-terminal region of tau, which is significant in neurodegenerative diseases such as Alzheimer's disease. Structurally, the epitope region can adopt an extended β-strand conformation, which may be linked to the seeding core in tau oligomers .

What applications is Phospho-MAPT (Ser404) Antibody validated for?

Based on comprehensive validation studies, Phospho-MAPT (Ser404) Antibody has been successfully used in multiple experimental applications:

Optimal dilutions should be determined by each laboratory for specific applications and conditions .

How should Phospho-MAPT (Ser404) Antibody be stored for maximum stability?

For optimal preservation of antibody activity:

• Long-term storage: Store at -20°C in small aliquots to avoid multiple freeze/thaw cycles

• Short-term use: Can be stored at 4°C

• Formulation: Typically supplied in PBS (pH 7.3-7.4), 150mM NaCl, with preservatives such as 0.02% sodium azide, and 50% glycerol as a stabilizer

A slight precipitate may form during storage at -20°C but can be dissolved by gentle vortexing without affecting antibody performance .

What is the specificity profile of Phospho-MAPT (Ser404) Antibody?

The specificity of the antibody has been rigorously characterized:

• The antibody detects tau only when phosphorylated at serine 404

• It shows reactivity with human, mouse, and rat samples

• Cross-reactivity studies have confirmed binding to phospho-tau in cellular lysates from these species

• Non-phospho specific antibodies are removed during purification by chromatography using non-phosphopeptide

Specificity can be validated by treating samples with lambda phosphatase, which should eliminate antibody reactivity .

How does the structural conformation of the pSer404 epitope influence antibody binding?

Crystal structure analyses of antigen-binding fragment (Fab)/epitope complexes reveal important conformational aspects:

The pSer404 epitope adopts a conformation remarkably similar to the pathogenic tau epitope pSer422, displaying a β-strand structure that may be linked to the seeding core in tau oligomers . This structural similarity is significant as both epitopes are associated with phosphorylation-dependent tau aggregation.

Different monoclonal antibodies (mAbs) that target the pSer404 region exhibit distinct binding modes:

• In the h4E6 antibody complex, six C-terminal residues 403TpSPRHL408 were observed, with the pSer404 side chain positioned toward the heavy chain

• The 8B2 and 6B2 antibodies have a different binding configuration, with the C-terminus of the peptide buried in the pocket at the center of the antigen-binding site

• Each of these mAbs has an antigen-binding pocket that accommodates the epitope from its C-terminal end, which differs from pSer396-specific antibodies where the epitope lies along the antigen-binding surface

This structural knowledge provides critical insights for designing more effective immunotherapeutic approaches against tau aggregation.

What is the role of Ser404 phosphorylation in the sequential phosphorylation mechanism of tau?

Ser404 phosphorylation plays a pivotal role in the sequential phosphorylation cascade of tau:

• Kinetic studies demonstrate that Ser404 is the first residue of tau to be efficiently phosphorylated by GSK3β, even when Ser214 is phosphorylated

• Ser404 phosphorylation is essential for subsequent phosphorylation by GSK3β, as mutation of Ser404 to alanine prevents all GSK3β activity on tau

• The phosphorylation follows a sequential pattern:

  • Initial priming phosphorylation at Ser404 (by CDK5 or other kinases)

  • GSK3β then sequentially phosphorylates Ser400

  • Finally, GSK3β phosphorylates Ser396

This sequential mechanism has significant implications for therapeutic targeting, as interrupting this cascade at the Ser404 stage could potentially prevent downstream hyperphosphorylation events associated with tau pathology.

How can Phospho-MAPT (Ser404) Antibody be used to distinguish pathological from physiological tau?

The ability to differentiate between normal and pathological tau is crucial for research and potential diagnostic applications:

• The Ser396/Ser404 region has received particular attention for therapeutic targeting because of its prominence and stability in diseased tissue

• These phosphorylation sites are hyperphosphorylated in Alzheimer's disease and other tauopathies

• Phosphorylation at Ser404 destabilizes microtubules, contributing to pathological processes

Methodological approach for distinguishing tau forms:

• Use Phospho-MAPT (Ser404) Antibody in combination with total tau antibodies

• Calculate the ratio of phosphorylated to total tau

• Compare phosphorylation patterns across multiple epitopes (pSer396, pSer404, pSer422)

• Analyze conformation-dependent epitopes that are exposed only in pathological tau aggregates

Research has shown that pSer404 immunoreactivity correlates with cognitive impairment and can be detected in early stages of disease progression .

What methodological considerations should be addressed when using Phospho-MAPT (Ser404) Antibody in competitive ELISA assays?

For researchers designing competitive ELISA experiments with Phospho-MAPT (Ser404) Antibody:

Two types of ELISA approaches have been validated:

• Standard solid phase assay: Binding of antibodies to peptides coated onto the plate

• Competition ELISA: Solution phase peptide competes with binding of the antibody to peptide coated on the plate

The competition ELISA clarifies how antibodies recognize peptides in solid phase versus solution, which can differ substantially. This is particularly important for phospho-epitopes where conformation plays a critical role in antibody recognition.

Methodological recommendations:

• Use peptides with different combinatorial phosphorylation arrangements of Ser396 and Ser404 to identify precise epitope specificity

• Include appropriate controls (non-phosphorylated peptide, peptides with adjacent phosphorylation sites)

• Consider the concentration gradient carefully (10-fold dilutions may miss optimal competition ranges)

• Account for potential conformational differences between solid-phase and solution-phase antigens

How does phosphorylation at Ser404 compare with other phosphorylation sites in tau for therapeutic targeting?

Research into tau immunotherapies provides important comparative data:

• The Ser396/Ser404 region is one of the most common approaches taken in clinical trials of patients with Alzheimer's disease

• pSer404 adopts a similar conformation to pSer422, another pathogenic epitope, suggesting common structural features in pathological tau

• Monoclonal antibodies targeting both pSer404 and pSer422 have shown therapeutic potential:

  • In pSer422-immunized animals, there is a decrease in aggregated tau and associated cognitive improvement

  • Similar effects have been observed with immunotherapies targeting pSer396/pSer404

An important methodological consideration is that tau protein is often C-terminally truncated, and residue 408 may be a terminus of some truncated tau forms. This allows certain monoclonal antibodies to react with tau paired helical filaments (PHF) isolated from human tissues .

What protocols should be followed for generating and validating phospho-specific antibodies against Ser404?

For researchers developing new phospho-specific antibodies:

• Immunogen design:

  • Use synthetic phosphopeptide conjugated to KLH (keyhole limpet hemocyanin)

  • The optimal peptide sequence should encompass the region around phosphorylation site of serine 404 (D-T-S(p)-P-R)

• Purification strategy:

  • Implement a two-step purification process:
    a) Affinity-chromatography using epitope-specific phosphopeptide
    b) Removal of non-phospho specific antibodies by chromatography using non-phosphopeptide

• Validation protocols:

  • ELISA with phosphorylated vs. non-phosphorylated peptides

  • Western blotting with phosphatase-treated controls

  • Immunohistochemistry comparing diseased vs. normal tissues

  • Competition assays with phosphorylated peptides

• Characterization of clones:

  • Select clones based on binding to the phospho-tau immunogen

  • Test reactivity against peptides with different phosphorylation patterns (pSer396 only, pSer404 only, pSer396/pSer404, non-phosphorylated)

The antibody generation process must be rigorous to ensure high specificity, as demonstrated in studies that produced monoclonal antibodies 8B2, 6B2, and 4E6 .

What controls should be included when using Phospho-MAPT (Ser404) Antibody in Western blot experiments?

A robust experimental design for Western blotting with Phospho-MAPT (Ser404) Antibody should include:

Essential controls:

• Positive control: Lysate from brain tissue known to contain hyperphosphorylated tau

• Negative control: Lambda phosphatase-treated sample to dephosphorylate tau

• Loading control: Total protein stain or housekeeping protein antibody

• Antibody specificity control: Pre-incubation of antibody with immunizing phosphopeptide

Recommended protocol adjustments:

• Sample preparation: Include phosphatase inhibitors in lysis buffer to preserve phosphorylation status

• SDS-PAGE conditions: Use 10-12% gels for optimal separation of tau isoforms (45-55 kDa range)

• Transfer conditions: Use PVDF membrane for better protein retention

• Blocking: 5% BSA in TBST is preferred over milk (which contains phosphatases)

• Antibody dilution: Start with 1:1000 for most applications, optimize as needed

How can researchers address inter-laboratory variability in Phospho-MAPT (Ser404) Antibody results?

Inter-laboratory variability is a common challenge in phospho-specific antibody applications. To minimize this:

• Standardize tissue processing:

  • Consistent fixation times for IHC samples (over-fixation can mask epitopes)

  • Standardized epitope retrieval methods

  • Uniform blocking protocols

• Consider preanalytical variables:

  • Post-mortem interval affects phosphorylation status

  • Rapid freezing of samples preserves phosphorylation state

  • Age of tissue samples impacts baseline phosphorylation

• Implement quantitative controls:

  • Use recombinant phosphorylated tau standards

  • Include reference tissue samples across experiments

  • Develop standard curves for each new antibody lot

• Technical recommendations:

  • Validate each new antibody lot with known positive controls

  • Share detailed protocols between laboratories

  • Consider round-robin testing of samples across laboratories for critical studies

What is the relationship between phosphorylation at Ser404 and other post-translational modifications of tau?

Understanding the interplay between different modifications is crucial for comprehensive tau research:

• Phosphorylation at Ser404 occurs within a S-P motif, targeted by proline-directed protein kinases including GSK3β and CDK5

• Phosphorylation decreases with age in normal conditions, but increases in pathological states

• The relationship with other modifications includes:

  Modification	Relationship with pSer404	Methodological Implication
  pSer396	Often co-phosphorylated, forms tandem epitope	Use dual phospho-antibodies for complete detection
  pSer422	Similar conformational structure	May cross-react in some assays
  Truncation	C-terminal truncation may remove Ser404	Use N-terminal antibodies in parallel
  Ubiquitination	May occur on nearby lysine residues	Check for shifted bands in Western blots
  Glycosylation	Can inhibit phosphorylation	Deglycosylation may increase signal

Researchers should consider using multiplexed approaches that detect multiple modifications simultaneously to gain a comprehensive view of tau's post-translational state.

How should researchers interpret discrepancies between different detection methods using Phospho-MAPT (Ser404) Antibody?

When faced with divergent results across different techniques:

• Consider method-specific limitations:

  • Western blot: Denatures proteins, may detect epitopes not accessible in native state

  • IHC/IF: Fixation can alter epitope accessibility

  • ELISA: May be affected by solution vs. solid-phase conformational differences

• Analytical approach to resolving discrepancies:

  • Perform epitope mapping to confirm exact binding sites

  • Compare results with other phospho-tau antibodies

  • Validate with phosphatase treatment controls

  • Consider using multiple antibody clones targeting the same epitope

• Interpretation framework:

  • Higher signals in denaturing methods suggest conformational masking in native state

  • Discrepancies between tissue and cell culture may reflect different kinase activities

  • Age-related differences in phosphorylation patterns must be accounted for

What are the implications of the β-strand conformation of the pSer404 epitope for tau aggregation and therapeutic targeting?

The structural studies revealing that pSer404 adopts a β-strand conformation have significant implications:

• Mechanistic insights:

  • This conformation is similar to pSer422, which is linked to the seeding core in tau oligomers

  • It supports the existence of proteopathic tau conformations stabilized by specific phosphorylation events

  • The extended β-strand structure may facilitate intermolecular interactions leading to aggregation

• Therapeutic considerations:

  • Antibodies recognizing this specific conformation may preferentially target pathological tau

  • Structure-guided optimization of antibodies could enhance binding to the β-strand conformation

  • The similar conformation between pSer404 and pSer422 suggests potential for antibodies with dual specificity

• Structural biology applications:

  • The crystal structures of three different monoclonal antibodies (8B2, 6B2, h4E6) bound to pSer404 provide templates for rational antibody design

  • These structures can inform computational modeling of tau aggregation

This structural knowledge creates an opportunity for immunological recognition of precise pathological species while minimizing binding to normal tau protein .

How can Phospho-MAPT (Ser404) Antibody contribute to the development of tau-targeted immunotherapies?

The Ser396/Ser404 region has emerged as a key therapeutic target:

• Therapeutic rationale:

  • This region shows prominence and stability in diseased tissue

  • Antibodies targeting this epitope could potentially block tau aggregation or promote clearance of pathological tau

• Preclinical evidence:

  • In animal models, immunization against the pSer396/pSer404 region has shown:

    • Decrease in aggregated tau

    • Associated cognitive improvement

  • Similar effects have been observed with immunotherapies targeting the conformationally similar pSer422 epitope

• Strategic considerations for therapeutic antibody development:

  • Focus on conformational specificity to target only pathological tau

  • Optimize blood-brain barrier penetration

  • Consider using humanized antibodies to reduce immunogenicity

  • Target extracellular tau to prevent spreading of pathology

• Emerging approaches:

  • Bispecific antibodies targeting multiple phospho-epitopes

  • Engineered fragments with enhanced brain penetration

  • Combinatorial approaches targeting tau and other pathological proteins

What novel methodologies are being developed for phosphorylation-specific tau detection beyond traditional antibody techniques?

Research is advancing beyond conventional antibody-based methods:

• Mass spectrometry approaches:

  • Quantitative MS for site-specific phosphorylation analysis

  • AQUA peptides for absolute quantification of phosphorylation stoichiometry

  • Phospho-proteomics for comprehensive mapping of tau modification patterns

• Biosensor technologies:

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • FRET-based sensors for conformational changes upon phosphorylation

  • Aptamer-based detection systems with phosphorylation specificity

• Single-molecule techniques:

  • Super-resolution microscopy to visualize tau aggregation states

  • Single-molecule FRET to detect conformational changes

  • Atomic force microscopy to characterize structural features

• Computational approaches:

  • Molecular dynamics simulations of phosphorylation effects on tau structure

  • Machine learning algorithms for predicting phosphorylation patterns

  • In silico screening for compounds that bind phosphorylated epitopes

These emerging technologies complement antibody-based methods and provide additional insights into tau phosphorylation dynamics and their role in disease progression.