Phospho-MAPT (S404) Recombinant Monoclonal Antibody

What is the Phospho-MAPT (S404) antibody and what does it specifically target?

The Phospho-MAPT (S404) antibody is a specialized immunological tool designed to recognize and bind to the microtubule-associated protein tau (MAPT) when it is phosphorylated specifically at the serine 404 position. This antibody enables researchers to detect a distinct post-translational modification of tau protein that has been implicated in the pathogenesis of neurodegenerative disorders, particularly Alzheimer's disease. The antibody binds with high specificity to the phosphorylated serine residue at position 404 in the tau protein sequence, allowing for precise detection of this modified form in experimental samples .

The specificity of this antibody is crucial because tau can be phosphorylated at multiple sites, and different phosphorylation patterns are associated with various pathological states. By using antibodies that target specific phosphorylation sites like S404, researchers can dissect the complex phosphorylation profile of tau in both normal and disease states .

What are the available forms of Phospho-MAPT (S404) antibodies and their sources?

Phospho-MAPT (S404) antibodies are available in several different forms to suit various research applications:

These antibodies are typically generated using synthetic phosphorylated peptides derived from the human MAPT sequence around the S404 position as immunogens . The polyclonal antibodies are produced in rabbits immunized with these peptides, while recombinant antibodies are generated through genetic engineering techniques where the antibody-encoding gene is incorporated into expression vectors and then transfected into host cells like HEK293F for production . After expression, the antibodies are purified using affinity chromatography methods to ensure high specificity and minimal cross-reactivity .

Why is the phosphorylation of tau at S404 significant in neurodegenerative disease research?

Research has shown that phosphorylation at S404 contributes to:

• Reduced binding of tau to microtubules, compromising microtubule stability and neuronal integrity

• Increased propensity for tau aggregation, promoting the formation of neurofibrillary tangles

• Altered tau distribution within neurons, contributing to the formation of neuropil threads in neurites

Notably, studies using mitochondrial inhibitors have demonstrated unique behaviors of different phospho-tau epitopes, with S404 phosphorylation showing distinct patterns compared to other sites. During mitochondrial stress, while many phospho-tau epitopes undergo rapid dephosphorylation, S404 phosphorylation can persist and show redistribution patterns associated with neuronal pathology . This suggests that S404 phosphorylation may play a specific role in the early stages of tau pathology in response to cellular stress.

How do different phosphorylation sites on tau, including S404, respond differently to mitochondrial dysfunction?

Research examining the effects of mitochondrial dysfunction on tau phosphorylation has revealed complex, epitope-specific responses that provide insights into potential early events in Alzheimer's disease pathogenesis. A study using chick primary neuron cultures treated with mitochondrial inhibitors demonstrated striking differences in the behavior of various phospho-tau epitopes .

Upon ATP reduction through mitochondrial inhibition:

• Epitopes recognized by antibodies AT8, AT270, AT180, S396, S404, and S422 showed progressive dephosphorylation over a 120-minute treatment period

• In contrast, epitopes recognized by the 12E8 antibody (which binds to phosphorylated KXGS motifs in the microtubule-binding domain) showed strong and sustained phosphorylation, with levels increasing to 232±69% at 60 minutes and maintaining elevated levels (155±4%) at 120 minutes

These findings suggest that different phosphorylation sites may drive distinct pathological processes during cellular stress, with some sites potentially playing more prominent roles in the early stages of disease pathogenesis.

What is the relationship between tau phosphorylation at S404 and actin-cofilin rod formation during cellular stress?

The relationship between tau phosphorylation at S404 and actin-cofilin (AC) rod formation represents an intriguing aspect of neuronal response to cellular stress that may have implications for early Alzheimer's disease pathology. Research using primary neuronal cultures has revealed a complex interplay between the cytoskeleton and tau phosphorylation following mitochondrial inhibition or actin depolymerization .

When neurons are subjected to mitochondrial stress:

• AC rods form within neurites as a stress response

• Different phospho-tau epitopes show distinct patterns of redistribution:

  • The 12E8 epitope (phosphorylated KXGS motifs in the microtubule-binding domain) predominantly localizes to rod-shaped AC aggregates

  • S404 phospho-tau (along with several other epitopes like S422 and AT270) can form spheroid inclusions, with rod-shaped structures observed less frequently

Importantly, experiments using Latrunculin B to induce actin depolymerization (without affecting ATP levels) demonstrated that:

• Actin depolymerization alone was sufficient to induce AC rod formation and recruitment of 12E8 epitopes

• This process did not significantly affect the distribution of other phosphoepitopes, including S404

These findings suggest that while S404 phosphorylation is affected during cellular stress, it may follow a different mechanistic pathway compared to the phosphorylation of KXGS motifs recognized by 12E8. The selective recruitment of certain phospho-tau epitopes to AC rods highlights a potential mechanism by which cytoskeletal abnormalities and tau pathology may be linked in the early stages of neurodegeneration .

How can Phospho-MAPT (S404) antibodies be used to distinguish between different stages of tau pathology in Alzheimer's disease?

Phospho-MAPT (S404) antibodies offer researchers a powerful tool for distinguishing between different stages of tau pathology in Alzheimer's disease by enabling the detection of specific phosphorylation patterns that evolve during disease progression. The temporal and spatial patterns of tau phosphorylation at S404 relative to other phosphorylation sites can provide insights into the sequence of pathological events .

In early-stage pathology:

• S404 phosphorylation may occur alongside other early phosphorylation events

• The distribution pattern remains relatively diffuse within neurons

• Co-localization with other phospho-tau epitopes may be limited

As pathology advances:

• S404 phosphorylated tau may redistribute into distinct aggregates

• These aggregates can form spheroid inclusions in neurites, potentially contributing to neuropil thread formation

• Co-localization with other pathological markers increases

By employing a panel of phospho-tau antibodies, including those targeting S404, researchers can conduct temporal mapping of tau phosphorylation events in experimental models and human tissue samples. This approach allows for the development of a phosphorylation signature that correlates with disease stages .

Additionally, the unique behavior of S404 phosphorylation during cellular stress conditions, particularly its redistribution pattern following mitochondrial inhibition, suggests it may serve as a marker for stress-induced tau alterations that precede more extensive aggregation. This makes Phospho-MAPT (S404) antibodies valuable tools for investigating the earliest stages of tau pathology when therapeutic intervention might be most effective.

What are the optimal protocols for using Phospho-MAPT (S404) antibodies in different experimental applications?

Optimizing protocols for Phospho-MAPT (S404) antibodies across different experimental applications requires careful consideration of multiple factors to ensure specific and reproducible results. Below are methodological guidelines for major applications:

Western Blot (WB):

• Sample preparation: Use phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and β-glycerophosphate) in lysis buffers to preserve phosphorylation states

• Recommended dilutions: 1:500 - 1:2000 for polyclonal antibodies like CABP0170

• Blocking: 5% BSA in TBST is preferable to milk, as milk contains phosphoproteins that may interfere with phospho-specific antibody binding

• Incubation: Overnight at 4°C is recommended for optimal sensitivity

• Secondary antibody: Anti-rabbit HRP conjugates typically at 1:5000 - 1:10000 dilution

Immunohistochemistry (IHC):

• Fixation: 4% paraformaldehyde is preferred for phospho-epitope preservation

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

• Recommended dilutions: 1:50 - 1:200 for most Phospho-MAPT (S404) antibodies

• Blocking: 10% normal serum from the species of the secondary antibody

• Detection systems: Both chromogenic (DAB) and fluorescent methods are suitable

Immunofluorescence/Immunocytochemistry (IF/ICC):

• Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilization: 0.1-0.3% Triton X-100 for 10 minutes

• Recommended dilutions: 1:100 - 1:200 for polyclonal antibodies

• Co-staining considerations: Compatible with neuronal markers (MAP2, NeuN) and other phospho-tau antibodies for co-localization studies

ELISA:

• Coating: 1-10 μg/ml of capture antibody or brain extract

• Blocking: 1-5% BSA in PBS

• Sample preparation: Include phosphatase inhibitors in extraction buffers

• Detection: Biotinylated secondary antibody followed by streptavidin-HRP

Optimization tips for all applications:

• Always include positive controls (e.g., brain tissue from Alzheimer's disease patients or SH-SY5Y cells )

• Include negative controls (non-phosphorylated samples) and phosphatase-treated samples to confirm specificity

• For reproducible results, standardize protein amounts across experiments and maintain consistent incubation times and temperatures

How can researchers validate the specificity of Phospho-MAPT (S404) antibodies in their experimental systems?

Validating the specificity of Phospho-MAPT (S404) antibodies is crucial for ensuring experimental rigor and reproducibility. Researchers should implement multiple complementary approaches to confirm that observed signals truly represent S404-phosphorylated tau rather than cross-reactivity or artifacts.

Comprehensive validation strategies include:

• Phosphatase treatment controls:

  • Treat duplicate samples with lambda phosphatase before immunoblotting or immunostaining

  • A specific phospho-antibody should show signal reduction or elimination after phosphatase treatment

• Peptide competition assays:

  • Pre-incubate the antibody with excess phosphorylated and non-phosphorylated peptides containing the S404 sequence

  • Signal should be blocked by the phosphorylated peptide but not by the non-phosphorylated version

• Knockout/knockdown controls:

  • Use MAPT knockout tissue/cells or MAPT siRNA knockdown samples as negative controls

  • Complete absence of signal confirms specificity for tau rather than cross-reactive proteins

• Correlation with other detection methods:

  • Compare results with other validated phospho-S404 antibodies from different sources

  • Use mass spectrometry to independently confirm the presence of phosphorylation at S404

• Site-directed mutagenesis:

  • Express wild-type tau and S404A mutant (serine to alanine) in cell models

  • Absence of signal with the S404A mutant confirms specificity for the phosphorylation site

• Induction experiments:

  • Treat samples with kinase activators known to increase S404 phosphorylation

  • Treatment with compounds like okadaic acid (phosphatase inhibitor) should enhance the signal

When validating results across multiple applications, researchers should also consider:

• Different fixation methods may affect epitope accessibility in IHC/ICC applications

• Denaturing conditions in Western blotting versus native conditions in IHC might affect antibody recognition

• The presence of other post-translational modifications near S404 might influence antibody binding

What are the key experimental considerations when studying phosphorylation dynamics at the S404 site in response to cellular stress?

Studying the phosphorylation dynamics at the S404 site of MAPT in response to cellular stress requires careful experimental design to capture the temporal and spatial changes that occur. Based on research findings, several critical experimental considerations should be addressed :

1. Time-course analysis:

2. Stress induction methods:

• Mitochondrial inhibitors (e.g., antimycin) affect multiple phosphorylation sites differently

• Actin depolymerizing agents (e.g., Latrunculin B) may have distinct effects on S404 versus other sites

• ATP measurement should be performed in parallel to correlate energy status with phosphorylation changes

3. Multi-epitope analysis:

• Always examine S404 phosphorylation in parallel with other phospho-tau epitopes (AT8, AT270, 12E8, etc.)

• Use total tau antibodies to normalize phosphorylation levels

• The ratio of different phospho-epitopes provides more informative data than absolute levels of a single epitope

4. Subcellular localization tracking:

• Combine biochemical assays (Western blot) with imaging techniques (immunofluorescence)

• Monitor redistribution into spheroid inclusions or rod-like structures

• Use co-localization studies with cytoskeletal markers (actin, cofilin) to assess recruitment to specific structures

5. Sample preparation considerations:

• Rapid fixation/lysis is critical as phosphorylation states can change during processing

• Include multiple phosphatase inhibitors in all buffers (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

• Separate analysis of soluble and insoluble tau fractions may reveal differential phosphorylation patterns

6. Quantification methods:

• For Western blots: normalize phospho-tau signal to total tau rather than housekeeping proteins

• For microscopy: measure both intensity and distribution patterns of phospho-S404 signal

• Advanced techniques like FRET-based biosensors can provide real-time monitoring of phosphorylation events

7. Correlation with functional outcomes:

• Assess microtubule binding capacity in parallel with phosphorylation changes

• Monitor neuronal viability and morphology alongside phosphorylation dynamics

• Evaluate downstream consequences like axonal transport efficiency

Research has shown that during mitochondrial stress, S404 phosphorylation exhibits distinct behavior, with redistribution into spheroid inclusions in neurites, sometimes forming rod-shaped structures. This pattern differs from other phospho-epitopes like those recognized by 12E8, which predominantly localize to actin-cofilin rods . These differences highlight the importance of comprehensive experimental approaches that can capture the complex and site-specific dynamics of tau phosphorylation during cellular stress.

What are the future research directions for Phospho-MAPT (S404) antibodies in neurodegenerative disease research?

The continued development and application of Phospho-MAPT (S404) antibodies offer promising avenues for advancing our understanding of neurodegenerative diseases, particularly Alzheimer's disease. Several key research directions emerge from current findings and technological capabilities:

Temporal mapping of tau pathology progression:
Future studies should focus on using Phospho-MAPT (S404) antibodies alongside other phospho-specific antibodies to establish a comprehensive temporal map of tau phosphorylation events during disease progression. This approach may identify critical windows for therapeutic intervention before irreversible neuronal damage occurs . The distinct behavior of S404 phosphorylation during cellular stress suggests it could serve as an early marker for pathological changes.

Mechanistic studies of phosphorylation regulation:
Research aimed at identifying the specific kinases and phosphatases that regulate S404 phosphorylation under normal and pathological conditions will provide valuable insights into disease mechanisms. The differential response of S404 phosphorylation to mitochondrial dysfunction compared to other phosphorylation sites suggests unique regulatory mechanisms that warrant further investigation .

Development of advanced diagnostic tools:
The specificity of Phospho-MAPT (S404) antibodies makes them candidates for developing more sensitive diagnostic assays for early-stage neurodegenerative diseases. Integration of these antibodies into multiplexed detection platforms could enable the creation of phosphorylation signatures that correlate with disease stages and progression rates .

Therapeutic target validation:
As potential therapeutic strategies aim to modulate tau phosphorylation, Phospho-MAPT (S404) antibodies will be crucial for validating target engagement and efficacy. Monitoring changes in S404 phosphorylation in response to experimental treatments could serve as a valuable biomarker for assessing therapeutic potential .

Integration with emerging technologies:
Combining Phospho-MAPT (S404) antibodies with advanced imaging techniques like super-resolution microscopy and expansion microscopy will enable more detailed visualization of the subcellular localization and interaction partners of phosphorylated tau. Additionally, incorporating these antibodies into single-cell analysis platforms could reveal cell-type-specific vulnerabilities in mixed neuronal populations .