Phospho-MAPT (S214) Antibody

What is Phospho-MAPT (S214) and why is it important in neuroscience research?

Phospho-MAPT (S214) refers to the microtubule-associated protein tau (MAPT) that has been specifically phosphorylated at the serine 214 residue. MAPT is a crucial protein that promotes microtubule assembly and stability, playing a vital role in maintaining neuronal cytoskeletal integrity and function . The phosphorylation at S214 is particularly significant as it modulates tau's affinity for microtubules and influences microtubule dynamics .

The importance of S214 phosphorylation stems from its specific functional consequences in neuronal contexts. According to current research, phosphorylation at Ser-214 by Serum and Glucocorticoid-regulated Kinase 1 (SGK1) mediates microtubule depolymerization and neurite formation in hippocampal neurons . This indicates that S214 phosphorylation serves as a regulatory switch for neuronal development and plasticity.

In neuroscience research, studying site-specific phosphorylation like S214 provides insights into how post-translational modifications regulate tau function in normal brain physiology and how dysregulation might contribute to neurodegenerative diseases characterized by tau pathology, such as Alzheimer's disease and other tauopathies.

What are the common applications of Phospho-MAPT (S214) antibodies in research?

Phospho-MAPT (S214) antibodies have multiple validated applications in neuroscience and protein biochemistry research:

Western Blotting (WB): These antibodies are widely used to detect and quantify S214-phosphorylated tau in protein lysates from cell cultures or tissue samples. Recommended dilution ranges typically vary from 1:500-1:2000, depending on the specific antibody and sample type .

Enzyme-Linked Immunosorbent Assay (ELISA): Provides quantitative measurement of phospho-tau levels in solution, allowing for high-throughput screening. Dilutions around 1:5000 are commonly recommended for this application .

Immunohistochemistry (IHC): Enables visualization of the distribution and abundance of S214-phosphorylated tau in tissue sections, which is particularly valuable for studying tau pathology in neurodegenerative diseases. Recommended dilutions for IHC typically range from 1:50-1:200 .

Dot Blot: Some antibodies, such as those from Abcam, have been validated for dot blot applications, providing a rapid method for detecting phospho-tau .

These applications have important research implications including:

• Investigation of disease mechanisms by examining how S214 phosphorylation changes in pathological conditions

• Drug discovery research through monitoring the effects of potential therapeutic compounds on tau phosphorylation

• Basic neurobiology studies to understand the role of S214 phosphorylation in normal neuronal function and development

As noted in the literature, phosphorylation state-specific antibodies like those targeting phospho-MAPT (S214) are "opening many exciting opportunities in investigative and diagnostic pathology," particularly for studying protein phosphorylation in situ .

What is the difference between monoclonal and polyclonal Phospho-MAPT (S214) antibodies?

Researchers can choose between monoclonal and polyclonal Phospho-MAPT (S214) antibodies, which differ in several important aspects that can impact experimental outcomes:

Polyclonal Phospho-MAPT (S214) Antibodies:

• Production: Generated by immunizing animals (typically rabbits) with a synthesized phosphopeptide derived from the region surrounding S214 of tau

• Epitope recognition: Contain a heterogeneous population of antibodies that recognize different epitopes on the phosphorylated tau protein

• Sensitivity: Generally offer broader epitope recognition, potentially increasing detection sensitivity

• Example: The rabbit polyclonal antibody from American Research Products recognizes phosphorylated tau at S214 with applications in ELISA and Western blot

Monoclonal Phospho-MAPT (S214) Antibodies:

• Production: Derived from a single B-cell clone, producing antibodies with identical binding properties

• Consistency: Provide superior lot-to-lot reproducibility

• Specificity: Higher specificity for a single epitope, reducing cross-reactivity

• Examples: Recombinant monoclonal antibodies such as those described by Cusabio and Abbexa

Methodological considerations when choosing between these types include:

• Experimental goals: Polyclonals may be preferred for applications requiring high sensitivity, while monoclonals offer advantages for studies requiring high specificity or where epitope conformational changes may occur.

• Reproducibility requirements: For longitudinal studies or experiments requiring consistent detection over time, monoclonals typically provide more consistent results.

• Cross-reactivity concerns: Monoclonals generally have lower cross-reactivity with similar phosphorylation sites.

The search results also highlight recombinant antibody technology as an advancement in antibody production. This approach involves "the insertion of the MAPT antibody-encoding gene into expression vectors," followed by transfection into host cells, allowing for standardized production without continuous animal immunization .

How can researchers validate the specificity of Phospho-MAPT (S214) antibodies?

Validating the specificity of Phospho-MAPT (S214) antibodies is essential for ensuring reliable experimental results. Researchers should employ several complementary techniques:

Phosphatase Treatment Controls:
The phospho-specific nature of these antibodies can be validated by treating samples with phosphatases to remove phosphate groups. A genuine phospho-specific antibody should show diminished or absent binding after phosphatase treatment.

Phosphorylation Site Mutants:
Creating tau constructs with S214 mutated to alanine (prevents phosphorylation) or to glutamic acid/aspartic acid (phospho-mimetic) allows for comparison of antibody reactivity . This approach provides strong evidence regarding epitope specificity.

Peptide Competition Assays:
Pre-incubating the antibody with the phosphopeptide used as the immunogen should block binding to phosphorylated tau in subsequent assays if the antibody is specific.

Cross-Reactivity Testing:
Testing the antibody against other phosphorylated proteins or tau phosphorylated at different sites helps establish specificity. A high-quality phospho-specific antibody "detects endogenous levels of Tau protein only when phosphorylated at S214" .

Multiple Application Validation:
Confirming antibody specificity across multiple applications strengthens confidence:

• "The Phospho-Mapt (S214) Recombinant Antibody has been validated for applications including ELISA and IHC"

• "Applications: ELISA, WB"

• "Following purification using affinity chromatography, the antibodies undergo testing through ELISA and IHC assays to confirm their recognition of the human MAPT protein phosphorylated at S214"

Comparison with Established Antibodies:
Comparing results with well-characterized antibodies targeting the same phosphorylation site provides another validation approach.

Species Cross-Reactivity Assessment:
Many Phospho-MAPT (S214) antibodies are tested for cross-reactivity with tau from different species:

• "Reactivity: Human, Mouse, Rat"

• "Reactivity: Human"

These validation approaches provide methodological frameworks to ensure antibodies specifically recognize tau phosphorylated at S214, minimizing the risk of false positives from cross-reactivity with other phosphorylation sites or proteins.

How should Phospho-MAPT (S214) antibodies be stored and handled to maintain their activity?

Proper storage and handling of Phospho-MAPT (S214) antibodies is critical for maintaining their specificity and sensitivity. Based on manufacturer recommendations:

Storage Temperature:
Most sources consistently recommend storing these antibodies at -20°C:

• "Store at -20°C for up to 1 year from the date of receipt"

• "Upon receipt, store at -20°C or -80°C"

• "Aliquot and store at -20°C"

Buffer Composition:
The antibodies are typically stored in a stabilizing buffer containing:

• Phosphate-buffered saline (PBS)

• Glycerol (typically 50%)

• Protein stabilizers like BSA (0.5%)

• Preservatives like sodium azide (0.02%)

Specific formulations include:

• "Liquid in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide"

• "PBS, pH 7.4, 150 mM NaCl, 0.02% sodium azide and 50% glycerol"

Avoiding Freeze-Thaw Cycles:
Multiple sources emphasize minimizing freeze-thaw cycles:

• "Avoid repeated freeze-thaw cycles"

• "Avoid repeat freeze"

Aliquoting Recommendation:
To prevent degradation from repeated freezing and thawing:

• "Aliquot and store at -20°C. Avoid repeated freeze/thaw cycles"

Methodological considerations for handling these antibodies include:

• Thawing Protocol: Thaw antibodies on ice or at 4°C rather than at room temperature to preserve activity.

• Working Dilutions: Prepare working dilutions fresh and avoid storing diluted antibody for extended periods.

• Temperature During Experiments: Maintain antibodies at appropriate temperatures during experiments (typically 4°C for incubations or room temperature for short procedures).

• Quality Control: Include positive controls in experiments to verify antibody activity, especially for older antibody stocks.

• Contamination Prevention: Use clean pipette tips and sterile techniques to prevent contamination which could degrade the antibody.

Following these storage and handling practices helps ensure the continued specificity and sensitivity of Phospho-MAPT (S214) antibodies for research applications.

What dilution ranges are recommended for different applications of Phospho-MAPT (S214) antibodies?

Optimal dilution ranges for Phospho-MAPT (S214) antibodies vary significantly depending on the specific application, antibody source, and experimental conditions. Based on manufacturer recommendations:

For Western Blotting (WB):

• Recommended range: 1:500-1:2000

For Enzyme-Linked Immunosorbent Assay (ELISA):

• Recommended dilution: 1:5000

For Immunohistochemistry (IHC):

• Recommended range: 1:50-1:200

These recommendations highlight that different applications require different antibody concentrations, with ELISA typically using the most dilute solutions and IHC requiring more concentrated antibody.

Methodological considerations for determining optimal dilutions include:

• Titration Experiments:
  It's generally advisable to perform initial titration experiments using a range of dilutions centered around the manufacturer's recommendations. This helps identify the optimal dilution for specific experimental conditions, which may vary depending on:

• Sample type (cell lysates, tissue sections, etc.)

• Target abundance

• Detection system sensitivity

• Background issues

• Signal-to-Noise Optimization:
  The goal is to find a dilution that maximizes specific signal while minimizing background. As noted by multiple suppliers, "Optimal dilutions/concentrations should be determined by the end user" .

• Antibody Source Considerations:
  Different manufacturers may produce antibodies with varying affinities and concentrations:

• Monoclonal antibodies may perform well at higher dilutions due to their specificity

• Polyclonal antibodies sometimes require lower dilutions (more concentrated solutions)

• Recombinant antibodies might have standardized affinities allowing for consistent dilution recommendations

• Experimental Variables Affecting Dilution:

• Incubation time: Longer incubations may allow for higher dilutions

• Incubation temperature: Higher temperatures might permit higher dilutions

• Detection system sensitivity: More sensitive detection systems allow for higher dilutions

When starting with a new antibody or application, it's prudent to test a range of dilutions spanning at least an order of magnitude around the manufacturer's recommendation to determine the optimal conditions for specific experimental setups.

How does phosphorylation at S214 affect tau protein function and microtubule binding?

Phosphorylation at S214 significantly alters tau protein function and its interaction with microtubules, with specific consequences for neuronal physiology:

Microtubule Dynamics Modulation:
According to current research, "Phosphorylation at Ser-214 by SGK1 mediates microtubule depolymerization and neurite formation in hippocampal neurons" . This indicates that S214 phosphorylation promotes microtubule disassembly, which is notable because tau's primary function is normally to stabilize microtubules. The phosphorylation essentially serves as a molecular switch to temporarily reduce tau's microtubule-stabilizing properties.

Affinity Regulation:
Phosphorylation at S214 "can modulate the affinity of MAPT for microtubules and influence microtubule dynamics" . This modulation of binding affinity represents a key mechanism for regulating the dynamic interaction between tau and microtubules, which is essential for proper neuronal function.

Neurite Formation:
The connection to neurite formation indicates that S214 phosphorylation plays a crucial role in neuronal development and plasticity . This suggests that controlled phosphorylation of tau at this site is part of the molecular machinery that guides neuronal growth and the establishment of connections.

Broader Context of Tau Phosphorylation:
Within the larger picture of tau regulation, "Phosphorylation within tau/MAP's repeat domain or in flanking regions seems to reduce tau/MAP's interaction with, respectively, microtubules or plasma membrane components" . While this statement doesn't specifically identify S214, it places this site within a general pattern where phosphorylation alters tau's interactions with cellular structures.

Kinase Specificity:
The enzyme responsible for this phosphorylation is clearly identified: "Phosphorylation at Ser-214 by SGK1 (Serum and Glucocorticoid-Regulated Kinase 1) mediates microtubule depolymerization and neurite formation in hippocampal neurons" . This identifies SGK1 as a key regulatory enzyme for tau function through its action at S214.

Research methodologies to investigate these effects typically include:

• In vitro microtubule binding assays comparing phosphorylated and non-phosphorylated tau

• Cell culture models expressing wild-type tau versus S214 phospho-mimetic mutants

• Neuronal cultures treated with compounds that activate or inhibit SGK1

• Immunocytochemistry using phospho-specific antibodies to correlate S214 phosphorylation with microtubule structures and neurite outgrowth

Understanding these relationships has significant implications for both basic neuroscience and for developing therapeutic strategies for tauopathies where phosphorylation patterns become dysregulated.

How does phosphorylation at S214 relate to other tau phosphorylation sites in tauopathies?

Tau phosphorylation at S214 exists within a complex network of phosphorylation events that together regulate tau function and contribute to pathology in neurodegenerative diseases:

Co-occurrence with Neighboring Phosphorylation Sites:
Recent research has characterized monoclonal tau antibodies "raised against a peptide in the PRR (208–225) with phosphorylation sites at Thr212, Ser214, Thr217" . This indicates that S214 is often studied in conjunction with nearby phosphorylation sites T212 and T217, suggesting these sites may work cooperatively in regulating tau function.

The same research noted that these antibodies "have demonstrated enhanced signal when sites T212, S214, and T217 are phosphorylated" , indicating that multiple phosphorylation events in this region may act synergistically.

Conformational Effects of Multiple Phosphorylations:
Evidence suggests that phosphorylation at S214, together with other sites, induces specific conformational changes in tau: "these antibodies are dependent upon conformational epitopes modulated by adjacent phosphorylation sites" . This indicates that phosphorylation patterns, rather than just individual sites, may drive tau structural changes relevant to pathology.

Experiments with "phospho-mimetic constructs" showed that "the presence of 12 phospho-mimetic residues just upstream of the core epitope region termed 12E greatly increased 5E2 and 2F12 binding" . This suggests that multiple phosphorylation events collectively influence tau conformation and antibody recognition in ways that single phosphorylation events do not.

Regulation and Cross-talk with Other Modifications:
There appears to be "reciprocal down-regulation of phosphorylation and O-GlcNAcylation" , indicating that tau phosphorylation (including at S214) exists in a complex relationship with other post-translational modifications like O-GlcNAcylation. This suggests intricate cross-talk between different types of protein modifications.

Pathological Relevance Across Multiple Tauopathies:
Phosphorylation patterns involving S214 appear relevant across different tau-related diseases. Antibodies sensitive to these phosphorylation patterns "label the neuropathological hallmarks of AD, PiD, CBD, and PSP" , suggesting that similar phosphorylation events occur across multiple tauopathies.

Methodological approaches to study these relationships include:

Understanding the relationships between different phosphorylation sites helps develop more comprehensive models of tau pathology and potentially identifies key events for therapeutic targeting in neurodegenerative diseases.

How can phospho-epitope lability affect the reliability of Phospho-MAPT (S214) antibody results?

Phospho-epitope lability represents a significant technical challenge when working with Phospho-MAPT (S214) antibodies that can substantially impact the reliability of experimental results:

Nature of the Challenge:
Phospho-epitope lability refers to the potential instability of phosphorylation modifications on proteins during sample preparation and analysis. As noted in the literature, "Technical considerations, including antibody specificity and lability of phosphoepitopes, are covered, along with potential pitfalls" . This instability can lead to false negative results or underestimation of phosphorylation levels.

Sources of Phospho-epitope Lability:

• Endogenous Phosphatases:
  Tissues and cells contain phosphatases that can rapidly dephosphorylate proteins once cells are disrupted during sample preparation, potentially eliminating the very epitopes the antibodies are designed to detect.

• Fixation Effects:
  Different fixation methods for tissues or cells (particularly for immunohistochemistry) can differentially affect phospho-epitope preservation.

• Post-mortem Interval:
  For human tissue samples, the time between death and tissue preservation can allow significant dephosphorylation due to continued phosphatase activity.

• Temperature Sensitivity:
  Phosphorylation marks can be temperature-sensitive, with higher temperatures potentially accelerating dephosphorylation.

Methodological Approaches to Address Phospho-epitope Lability:

• Phosphatase Inhibitors:
  Including comprehensive phosphatase inhibitors in all buffers used during sample preparation is essential. Common inhibitors include:

• Sodium fluoride (NaF)

• Sodium orthovanadate (Na₃VO₄)

• β-glycerophosphate

• Commercial phosphatase inhibitor cocktails

Impact on Research Interpretation:

Failure to address phospho-epitope lability can lead to:

• False negatives (inability to detect genuinely phosphorylated tau)

• Inconsistent results between experiments

• Inability to detect subtle differences in phosphorylation levels

• Misinterpretation of regional or cellular phosphorylation patterns

Understanding and controlling for phospho-epitope lability is therefore crucial for generating reliable and reproducible results with Phospho-MAPT (S214) antibodies in neurodegenerative disease research.

How do conformation-dependent Phospho-MAPT (S214) antibodies detect structural changes in tau?

Recent advances have led to the development of conformation-dependent antibodies that recognize phosphorylated tau, including at the S214 position. These specialized antibodies provide unique insights into tau structural changes:

Recognition Mechanism:
Unlike strictly phospho-specific antibodies that primarily recognize the phosphate group and surrounding amino acids, conformation-dependent antibodies recognize three-dimensional epitopes that form when tau adopts specific structural conformations.

Recent research has characterized "two monoclonal tau antibodies raised against a peptide in the PRR (208–225) with phosphorylation sites at Thr212, Ser214, Thr217. These antibodies (which we refer to as 5E2 and 2F12) have demonstrated enhanced signal when sites T212, S214, and T217 are phosphorylated, but are not completely dependent on phosphate presence" .

The critical finding was that "these antibodies are dependent upon conformational epitopes modulated by adjacent phosphorylation sites" . This indicates they detect structural features that emerge when tau is phosphorylated at specific patterns.

Experimental Approaches to Study These Effects:

• Phospho-mimetic Mutations:
  "The properties of the 5E2 and 2F12 antibodies were further characterized by expressing WT tau and phospho-mimetic constructs, with residues altered both upstream and downstream of the core epitopes" .

• Sequential Mutation Analysis:
  "Breaking down these 12 phospho-mimetic residues into a series of four mutations termed 4E(198–205), 4E(231–238), and 4E(210–217) revealed that this enhanced binding is due to the latter set of four phospho-mimetic residues" .

Research Applications:

• Pathological Conformations:
  These antibodies can detect disease-specific conformations of tau: "these antibodies label the neuropathological hallmarks of AD, PiD, CBD, and PSP" .

• Disease-Specific Tau Conformations:
  They may help distinguish between different tauopathies based on specific conformational signatures.

• Drug Development:
  These antibodies could be valuable for screening drugs designed to prevent pathological tau conformational changes.

• Disease Progression Monitoring:
  Changes in the abundance of specific tau conformations could serve as biomarkers for disease progression.

Methodological Considerations:

When using conformation-dependent antibodies:

• Sample preparation methods must preserve native protein conformations, which may require milder conditions than those used for strictly sequence-dependent epitopes.

• Results should be interpreted in the context of both phosphorylation status and protein conformation.

• Combining conformation-dependent antibodies with strictly phospho-specific antibodies can provide more complete information about both modifications and resulting structural changes.

These specialized antibodies represent advanced tools for studying the complex relationships between tau phosphorylation, conformation, and pathology in neurodegenerative diseases.