Phospho-SYT1/SYT2 (Thr202/199) Antibody

What is the specific target and epitope of Phospho-SYT1/SYT2 (Thr202/199) antibody?

This antibody specifically detects endogenous levels of Synaptotagmin 1 (SYT1) and Synaptotagmin 2 (SYT2) proteins only when phosphorylated at threonine 202 (for SYT1) or threonine 199 (for SYT2). The epitope is centered around the phosphorylated threonine residue with the specific sequence R-K-T(p)-L-N derived from human Synaptotagmin . This antibody recognizes the critical phosphorylation site that regulates synaptic vesicle trafficking and neurotransmitter release. The antibodies are typically produced by immunizing rabbits with synthetic phosphopeptide and KLH conjugates, then purified by affinity-chromatography using epitope-specific phosphopeptide .

What are the validated applications and recommended dilutions for this antibody?

The Phospho-SYT1/SYT2 (Thr202/199) antibody has been validated for multiple research applications with specific recommended dilutions:

The antibody has demonstrated reactivity in human, mouse, and rat specimens, with predicted cross-reactivity in pig, bovine, horse, sheep, rabbit, dog, chicken, and Xenopus based on sequence homology .

How should I validate the specificity of this phospho-specific antibody?

Proper validation of phospho-specific antibodies is critical for research reliability. For Phospho-SYT1/SYT2 (Thr202/199) antibody, implement the following validation strategy:

• Alkaline phosphatase (AP) treatment: This serves as an essential negative control. Samples treated with alkaline phosphatase should show significantly reduced or abolished signal, confirming phospho-specificity .

• Non-phosphopeptide controls: Use a parallel antibody purification technique where non-phospho specific antibodies are removed by chromatography using non-phosphopeptide .

• Western blot validation: Independent verification showing a single band at the expected molecular weight (approximately 60 kDa observed) indicates specificity. According to reverse phase protein array (RPPA) studies, approximately 85% of phospho-antibodies showing expected responses to AP treatment demonstrate meaningful single bands at expected sizes in western blots .

• Genetic controls: Testing on SYT1/SYT2 knockout tissues or cells, or using phospho-mutants (e.g., T112A mutation, equivalent to T202A) should show no signal .

• Peptide competition assay: Pre-incubating the antibody with excess phospho-peptide should abolish specific signal in western blots .

What is the biological significance of SYT1/SYT2 phosphorylation at Thr202/199?

Phosphorylation of SYT1 at Thr202 (or the equivalent Thr199 in SYT2) plays several crucial roles in neurotransmission:

• Post-priming regulation: Phosphorylation at this site controls a post-priming step in synaptic vesicle release, positioning this modification as a key regulator of neurotransmitter release efficiency .

• PKC-dependent potentiation: Research shows that phosphorylation at this site is essential for PKC-dependent potentiation of evoked neurotransmitter release. Studies demonstrate that in neurons expressing the phospho-deficient Syt T112A mutant (equivalent to T202A), phorbol myristate acetate (PMA) application fails to potentiate evoked excitatory postsynaptic currents (EPSCs), whereas wild-type SYT1 supports approximately 40% potentiation .

• Synapse-specific plasticity: This phosphorylation enhances potentiation after high-frequency stimulation (HFS). In experiments using 200 action potentials at 100 Hz, SYT1 wild-type expressing neurons showed potentiation in 42% of cells, while neurons expressing phospho-deficient SYT1 showed potentiation in only 7.7% of cells .

• Regulation of membrane interactions: Phosphorylation may alter the interaction of SYT1/SYT2 with phospholipid membranes during trafficking of synaptic vesicles at the active zone of the synapse .

How do SYT1 and SYT2 differ in structure and function regarding this phosphorylation site?

Despite their high sequence homology, SYT1 and SYT2 have notable differences relevant to the Thr202/199 phosphorylation site:

• Sequence difference: SYT2 lacks seven amino acid residues within the linker between the transmembrane domain (TM) and the C2A domain, where the PKC/CaMK-II phosphorylation site in SYT1 resides .

• Tissue expression: SYT1 is predominantly expressed in rostral, phylogenetically younger brain regions, while SYT2 shows a different distribution pattern across the nervous system .

• Response to phorbol esters: Despite the structural differences, SYT2-dependent synapses still show normal phorbol-ester-induced potentiation. When Syt1 knockout cells were rescued with Syt2 or Syt1 with identical deletion (Syt1 Δ109–116), both groups showed rapid and prominent facilitation upon addition of phorbol esters that actually exceeded potentiation seen with wild-type SYT1 .

• Inhibitory role of SYT1 linker: The seven amino acid sequence within the linker of SYT1 appears to have an inhibitory role at rest and becomes permissive of phorbol-ester-induced potentiation upon phosphorylation of T112 (equivalent to T202) .

What are the optimal experimental protocols for detecting phospho-SYT1/SYT2 in different neural preparations?

Different neural preparations require specific protocols for optimal phospho-SYT1/SYT2 detection:

For cultured neurons (Immunofluorescence/ICC):

• Fix cells with paraformaldehyde (PFA) and permeabilize with 0.1% Triton X-100

• Block in 10% serum for 45 minutes at 25°C

• Apply primary antibody at 1:200 dilution and incubate for 1 hour at 37°C

• Detect with appropriate secondary antibody (e.g., Alexa Fluor 594 conjugated goat anti-rabbit IgG) at 1:600 dilution

• Include phosphatase inhibitors throughout the procedure to prevent dephosphorylation

For tissue sections (IHC):

• For formalin-fixed paraffin-embedded (FFPE) tissues, perform heat-mediated antigen retrieval in citrate buffer

• Block tissue and incubate with primary antibody (1:50-1:200) for 1.5 hours at 22°C

• Use HRP-conjugated secondary antibody for detection

• For reverse phase protein array (RPPA) applications, validate antibody performance with both phosphatase-treated and untreated samples

For biochemical analysis (Western Blot):

• Include phosphatase inhibitors in lysis buffers

• Run paired samples (±phosphatase treatment) to confirm specificity

• Use 1:500-1:2000 dilution range for primary antibody

• Consider running a mobility shift assay, as phosphorylated forms may run at a slightly higher apparent molecular weight

How can I correlate phospho-SYT1/SYT2 with functional synaptic changes in electrophysiological experiments?

To establish meaningful correlations between phospho-SYT1/SYT2 levels and synaptic function:

• Patch-clamp recording with post-hoc immunostaining:

  • Perform whole-cell recordings on identified neurons

  • Apply specific stimulation protocols known to induce phosphorylation (e.g., high-frequency stimulation at 100 Hz)

  • Fix cells immediately after recording

  • Perform immunostaining for phospho-SYT1/SYT2

  • Include biocytin or Lucifer Yellow in the recording pipette to allow for post-hoc identification

• Rescue experiments with phospho-mutants:

  • Use Syt1 knockout neurons

  • Rescue with either wild-type SYT1, phospho-null (T112A/T202A), or phospho-mimetic (T112D/T202D) variants

  • Compare evoked EPSCs, paired-pulse facilitation, and response to high-frequency stimulation

  • Research has shown that neurons expressing SYT1 T112A fail to show PMA-induced potentiation of evoked release while maintaining PMA-induced increases in spontaneous release

• Pharmacological manipulations:

  • Apply PKC activators (PMA, PDBu at 1 μM) to induce phosphorylation

  • Use PKC inhibitors to block phosphorylation

  • Document changes in both phosphorylation status and synaptic parameters

  • Example: PMA increases EPSC amplitude by ~40% in SYT1 WT-expressing cells but has no effect on EPSC amplitude in SYT1 T112A-expressing cells

• High-frequency stimulation protocols:

  • The induction protocol of 200 action potentials at 100 Hz reveals differences between wild-type and phospho-mutant SYT1

  • In SYT1 wild-type rescued cells, 42% showed potentiation after HFS

  • In SYT1 T112A rescued cells, only 7.7% showed potentiation

What are the upstream kinases responsible for SYT1/SYT2 phosphorylation, and how can I manipulate these pathways?

The primary kinases that phosphorylate SYT1 at Thr202 include:

• Protein Kinase C (PKC):

  • Activated by diacylglycerol (DAG) and calcium

  • Can be experimentally activated with phorbol esters like PMA or PDBu (typically at 1 μM concentration)

  • Inhibited by compounds like GF109203X or Gö6983

  • Works in conjunction with other proteins in the DAG/PKC pathway including Munc13 and Munc18-1

• Ca²⁺/calmodulin-dependent protein kinase II (CaMKII):

  • Activated by calcium influx

  • Can be inhibited by KN-93

  • May work alongside PKC in activity-dependent phosphorylation

For experimental manipulation:

• Activate PKC-dependent phosphorylation with PMA or PDBu (1 μM)

• Activate pathways that increase intracellular calcium to stimulate both PKC and CaMKII

• Use high-frequency stimulation (HFS) protocols (e.g., 200 APs at 100 Hz) to physiologically induce phosphorylation

• Block calcium influx through NMDA receptors or voltage-gated calcium channels to prevent activity-dependent phosphorylation

• Consider manipulating the Mid1-dependent high-affinity Ca²⁺ influx system, which may contribute to activation of calcium-dependent kinases

How do I best design experiments to distinguish the effects of phospho-SYT1/SYT2 from other phosphoproteins in synaptic plasticity?

To isolate the specific contribution of phospho-SYT1/SYT2:

• Use of phospho-mutants:

  • Express SYT1 T112A (phospho-null) or T112D (phospho-mimetic) in SYT1 knockout backgrounds

  • Compare with wild-type rescue to isolate phosphorylation-specific effects

  • Research shows that SYT1 T112D fully supports potentiation of both spontaneous and evoked release, similar to wild-type SYT1

• Combined mutations approach:

  • Studies indicate that phosphorylation of both SYT1 and Munc18-1 is required for potentiation of evoked release, whereas phosphorylation of a single substrate is not sufficient

  • Create experimental conditions where only SYT1 can be phosphorylated (e.g., using Munc18-1 phospho-null mutants) to isolate its specific contribution

• Temporal dynamics analysis:

  • Different phosphoproteins may show distinct temporal patterns of phosphorylation

  • Track phosphorylation over time after stimulation to identify protein-specific signatures

  • Compare with electrophysiological measures at matched time points

• Triple knockout approaches:

  • Use sophisticated genetic models like Doc2a/Doc2b/Syt1 triple knockout (TKO) mice

  • These models have revealed that in the absence of SYT1 phosphorylation, spontaneous release potentiation is greatly reduced but not completely abolished, suggesting partial redundancy with Doc2 proteins

• Pathway-specific interventions:

  • The DAG/PKC pathway influences multiple proteins including Munc13, PKC, Munc18-1, and SYT1

  • Different forms of plasticity have different dependencies:

    • Paired-pulse plasticity depends on Munc13-1, PKC, and Munc18-1 phosphorylation, but SYT1 phosphorylation is dispensable

    • Potentiation by direct DAG application depends on all these factors

    • HFS-induced potentiation requires PKC activation, Munc18-1 phosphorylation, and SYT1 phosphorylation

What are the methodological challenges in detecting phospho-SYT1/SYT2 in post-mortem tissue samples?

Detecting phospho-SYT1/SYT2 in post-mortem samples presents several challenges:

• Rapid post-mortem dephosphorylation:

  • Continued phosphatase activity after death can rapidly dephosphorylate proteins

  • Include phosphatase inhibitors during tissue collection and processing

  • Document post-mortem interval (PMI) and control for this variable when comparing samples

  • Consider flash-freezing samples immediately after collection

• Epitope masking and retrieval:

  • Formalin fixation can mask phospho-epitopes

  • Utilize optimized antigen retrieval methods for FFPE tissues

  • Heat-mediated antigen retrieval in citrate buffer is recommended for this antibody

  • Compare FFPE with fresh frozen (FF) tissue preparation methods, as concordance rates vary for different phospho-antibodies (approximately 40% for many antibodies)

• Validation strategies for post-mortem tissue:

  • Include positive controls from fresh tissue samples

  • Use alkaline phosphatase treatment as a negative control

  • Include region-specific controls, as SYT1 and SYT2 show differential expression across brain regions

  • Consider western blot validation of specific post-mortem samples before immunohistochemistry

• Technical adaptations:

  • Increase antibody concentration for post-mortem tissue (starting at the higher end of the recommended range)

  • Extend incubation times to improve penetration

  • Consider tyrosine phosphatase application as a negative control for phospho-peptide characterization

  • Utilize fluorescence-based detection approaches and check signal-to-background ratio, spot quality, and reproducibility

How can I integrate phospho-SYT1/SYT2 studies with broader investigations of synaptic phosphoproteomes?

To position phospho-SYT1/SYT2 studies within the broader synaptic phosphoproteome:

• Mass spectrometry-based approaches:

  • Perform phosphoproteomic analysis of synaptic fractions under various stimulation conditions

  • Identify co-regulated phosphorylation sites that change in parallel with SYT1/SYT2

  • Use RPPA (Reverse Phase Protein Array) for high-throughput profiling of phospho-proteins

  • Validate mass spectrometry findings with antibody-based methods for specific proteins

• Pathway analysis:

  • Map phosphorylation events in the DAG/PKC pathway

  • Determine the sequence of phosphorylation events following stimulation

  • Establish causal relationships between different phosphorylation events

  • Consider that phosphorylation of both SYT1 and Munc18-1 is required for potentiation of evoked release, indicating pathway integration

• Multi-protein phosphorylation studies:

  • Simultaneously monitor phosphorylation of SYT1/SYT2, Munc18-1, and other known phosphoproteins

  • Use multiplexed western blotting or immunofluorescence techniques

  • Correlate changes with functional readouts of synaptic transmission

  • Analyze how different phosphorylation events might cooperate in regulating distinct aspects of neurotransmitter release:

    • RRP refilling after 40-Hz stimulation depends on Munc13-1, PKC, and Munc18-1 phosphorylation, but SYT1 phosphorylation is dispensable

    • Different phosphoproteins may regulate different stages of the synaptic vesicle cycle

• Temporal dynamics differentiation:

  • Different phosphoproteins exhibit distinct temporal profiles of phosphorylation and dephosphorylation

  • Design time-course experiments to differentiate rapid versus sustained phosphorylation events

  • Correlate these temporal profiles with electrophysiological measurements of synaptic function at matching time points