Phospho-SYT1 (Ser309) Antibody

What is Synaptotagmin-1 and what is the significance of its phosphorylation at Ser309?

Synaptotagmin-1 (SYT1) is a ~60-62 kDa synaptic vesicle protein localized in synaptic vesicles and chromaffin granules, serving as a key calcium sensor for neurotransmitter release . Phosphorylation at Ser309 represents one of several regulatory post-translational modifications that can significantly alter synaptotagmin's functional properties . This specific phosphorylation site may play a critical role in modulating the protein's ability to influence exocytosis and endocytosis processes during synaptic transmission.

The modification occurs within a functionally important region of the protein and can influence synaptotagmin's interactions with lipid membranes and other synaptic proteins. SYT1 binds acidic phospholipids with specificity requiring both an acidic head group and a diacyl backbone, a property potentially regulated by phosphorylation states .

What experimental applications are recommended for Phospho-SYT1 (Ser309) antibodies?

Based on validated research protocols, Phospho-SYT1 (Ser309) antibodies have demonstrated efficacy in several experimental applications:

• Western Blot (recommended dilution ~1:1000): Effective for detecting the ~60-62 kDa phosphorylated SYT1 protein in tissue lysates

• Immunohistochemistry (recommended dilution ~1:400): Useful for visualizing the spatial distribution of phosphorylated SYT1 in tissue sections

• Immunocytochemistry/Immunofluorescence (recommended dilution ~1:100-1:400): Enables subcellular localization studies of phosphorylated SYT1

• ELISA: Appropriate for quantitative assessment of phosphorylated SYT1 levels

When designing experiments, researchers should consider that phosphorylation is labile and may require special sample handling procedures to maintain integrity.

How can I validate the specificity of a Phospho-SYT1 (Ser309) antibody?

A multi-faceted approach to antibody validation is recommended:

• Lambda phosphatase treatment: Treating samples with lambda phosphatase prior to immunoblotting should eliminate or dramatically reduce signal if the antibody is truly phospho-specific. This has been demonstrated with Phospho-SYT1 (Ser309) antibodies where immunolabeling was completely eliminated after phosphatase treatment .

• Positive and negative controls: Use samples with known phosphorylation status (e.g., stimulated versus unstimulated neuronal preparations).

• Peptide competition: Pre-incubation of the antibody with the phosphorylated peptide immunogen should block specific binding.

• Cross-species reactivity testing: Validate reactivity across your species of interest, as the epitope surrounding Ser309 is highly conserved across many species including human, rat, mouse, bovine, canine, chicken, primate, and zebrafish .

What are the optimal experimental conditions for detecting Phospho-SYT1 (Ser309) in different neural tissue preparations?

Optimal experimental conditions vary by preparation type and must balance signal preservation with technical requirements:

For cultured neurons:

• Fixation: Brief (10-15 min) 4% paraformaldehyde fixation is typically sufficient

• Permeabilization: Gentle detergent treatment (0.1% Triton X-100)

• Blocking: 3-5% BSA or serum that does not cross-react with the primary antibody

• Antibody incubation: Overnight at 4°C at dilutions of 1:100-1:400 for immunofluorescence

For tissue sections:

• Fresh-frozen or perfusion-fixed tissue may be used, though phospho-epitopes are often better preserved in fresh-frozen specimens

• Antigen retrieval may be necessary but should be gentle to preserve phosphorylation

• Recommended antibody dilution of approximately 1:400 for immunohistochemistry

For Western blotting:

• Sample preparation must include phosphatase inhibitors

• Protein extraction buffers should be kept cold and contain EDTA

• Samples should be processed quickly to prevent dephosphorylation

• Recommended antibody dilution of approximately 1:1000

Critical considerations for all preparations:

• Include phosphatase inhibitor cocktails in all buffers

• Maintain cold temperatures during sample preparation

• Process samples quickly to minimize dephosphorylation

How does Phospho-SYT1 (Ser309) interact with other synaptic proteins and what techniques best demonstrate these interactions?

Phosphorylation of SYT1 at Ser309 potentially modulates its interactions with several binding partners. While the search results don't specifically detail all partners affected by Ser309 phosphorylation, we can infer connections from related research:

Interaction partners potentially affected by phosphorylation:

• SNARE complex proteins (particularly syntaxin)

• AP2 adaptor complex

• Neurexins

• SV2A (Synaptic Vesicle Protein 2A)

Research indicates that phosphorylation of SV2A at Thr84 controls its interaction with synaptotagmin-1, suggesting a phosphorylation-dependent interaction network between these proteins . This hints at a broader regulatory mechanism where phosphorylation status of multiple synaptic proteins coordinates vesicle trafficking.

Techniques to demonstrate these interactions:

• Co-immunoprecipitation with phospho-specific antibodies

• Proximity ligation assay (PLA) to visualize protein interactions in situ

• FRET/FLIM imaging using tagged constructs

• Pull-down assays with phosphomimetic mutants (S309D) versus phospho-deficient mutants (S309A)

• Cross-linking mass spectrometry to identify interaction interfaces

A combination approach comparing wild-type, phosphomimetic, and phospho-deficient mutants can reveal how phosphorylation at Ser309 modulates protein-protein interactions in the synaptic vesicle cycle.

What are the known kinases that phosphorylate Synaptotagmin-1 at Ser309 and how can this phosphorylation be manipulated experimentally?

While the specific kinases targeting Ser309 are not explicitly detailed in the search results, SYT1 is known to be phosphorylated by multiple protein kinases . Based on the sequence context, likely candidate kinases include:

• Casein kinase 1 (CK1) family members - research has shown CK1 family involvement in synaptic protein phosphorylation

• Casein kinase 2 (CK2)

• Protein kinase C (PKC)

• Calcium/calmodulin-dependent protein kinase II (CaMKII)

Experimental manipulation approaches:

• Pharmacological:

  • Kinase inhibitors specific to candidate kinases

  • Phosphatase inhibitors to maintain phosphorylation

  • Calcium modulators to affect calcium-dependent kinases

• Genetic:

  • Expression of phosphomimetic (S309D/E) or phosphodeficient (S309A) mutants

  • Knockdown/knockout of candidate kinases

  • Overexpression of specific protein kinases

• Stimulation protocols:

  • High-frequency neuronal stimulation protocols

  • Depolarization with KCl or field stimulation

  • Activation of specific signaling pathways (e.g., PKC activation with phorbol esters)

A systematic approach combining these methods can help establish the regulatory kinases and signaling pathways governing Ser309 phosphorylation.

What are common challenges when working with Phospho-SYT1 (Ser309) antibodies and how can they be overcome?

Challenge 1: Loss of phosphorylation during sample preparation

• Solution: Always include phosphatase inhibitor cocktails in all buffers

• Maintain samples at 4°C during processing

• Process samples rapidly to minimize dephosphorylation

• Use fresh samples whenever possible

Challenge 2: Weak or inconsistent signal

• Solution: Optimize antibody concentration (typical working dilutions range from 1:100-1:1000 depending on application)

• Try different blocking buffers (BSA vs. serum)

• Extend primary antibody incubation time (overnight at 4°C)

• Consider different detection systems for enhanced sensitivity

Challenge 3: Background or non-specific staining

• Solution: Increase blocking time and concentration

• Perform more stringent washing steps

• Pre-adsorb antibody with non-specific proteins

• Validate specificity through lambda phosphatase treatment

Challenge 4: Cross-reactivity with other phosphorylated proteins

• Solution: Perform peptide competition controls

• Use phospho-deficient mutants as negative controls

• Verify molecular weight in Western blots (~60-62 kDa)

• Compare results with a second antibody targeting a different epitope on SYT1

Challenge 5: Variability between antibody lots

• Solution: Test each new lot against a standard sample

• Maintain consistent experimental conditions

• Consider generating a large batch of positive control lysate

How can phosphatase treatments be effectively used as controls in Phospho-SYT1 (Ser309) experiments?

Phosphatase treatment serves as a critical control to validate phospho-specific antibodies:

Protocol for lambda phosphatase treatment:

• Prepare two identical samples of your protein extract

• Treat one sample with lambda phosphatase (typical protocol: 1200 units for 30 minutes at 30°C)

• Leave the other sample untreated (control)

• Run both samples on Western blot and probe with the Phospho-SYT1 (Ser309) antibody

Expected results:

• The untreated control should show the ~60-62 kDa band corresponding to phosphorylated SYT1

• The phosphatase-treated sample should show dramatically reduced or eliminated signal

• This difference confirms the phospho-specificity of the antibody

Important considerations:

• Include appropriate buffers and cofactors for optimal phosphatase activity

• Consider time-course experiments to determine optimal treatment duration

• For tissue sections or fixed cells, phosphatase treatment needs to be performed before fixation

• Always prepare fresh phosphatase solutions

• Include controls for phosphatase effectiveness (known phosphoprotein)

What are the best sample preparation methods to preserve phosphorylation at Ser309?

Preserving phosphorylation requires attention to several key factors:

For tissue extraction:

• Rapidly harvest and flash-freeze tissue in liquid nitrogen

• Use extraction buffer containing:

  • Strong phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Protease inhibitor cocktail

  • EDTA to chelate divalent cations required for phosphatase activity

  • Gentle detergents (e.g., 1% NP-40 or 0.5% Triton X-100)

• Maintain cold temperature (4°C) throughout extraction

• Process samples quickly (avoid freeze-thaw cycles)

For cultured cells:

• Wash rapidly with ice-cold PBS containing phosphatase inhibitors

• Lyse cells directly in sample buffer containing phosphatase inhibitors

• Alternatively, scrape cells into phosphatase inhibitor-containing buffer

For immunohistochemistry/immunocytochemistry:

• Consider using in vivo fixation (perfusion) with fixatives containing phosphatase inhibitors

• Minimize time between tissue harvest and fixation

• Use gentle fixation protocols (overfixation can mask epitopes)

• For cultured neurons, rapid fixation with pre-warmed 4% paraformaldehyde

Storage recommendations:

• Store antibodies at -20°C for long-term storage

• For frequent use, small aliquots may be stored at 4°C for up to one month

• Avoid repeated freeze-thaw cycles

• Store lysates at -80°C with phosphatase inhibitors

How is Phospho-SYT1 (Ser309) being used in current neuroscience research?

Phospho-SYT1 (Ser309) antibodies are valuable tools in several areas of neuroscience research:

Synaptic vesicle cycling studies:

• Investigating the role of phosphorylation in regulating exocytosis and endocytosis

• Tracking activity-dependent changes in phosphorylation status

• Studying the temporal dynamics of phosphorylation during different phases of neurotransmission

Neuronal development research:

• Examining how phosphorylation states change during synapse formation and maturation

• Investigating the role of SYT1 phosphorylation in dendrite formation, as SYT1 plays a role in this process

Relationship with other synaptic proteins:

• Studying phosphorylation-dependent interactions with SV2A and other synaptic proteins

• Investigating the "phospho-switch" mechanisms in synaptic vesicle recycling

Stimulus-response coupling:

• Examining how neuronal activity regulates SYT1 phosphorylation

• Investigating the temporal relationship between calcium influx and phosphorylation events

Dual-color immunofluorescence applications:

• Studies combining Phospho-SYT1 (Ser309) antibodies with markers like PSD95 allow visualization of phosphorylated synaptotagmin in relation to postsynaptic structures, as demonstrated in cortical neuron studies

What is the relationship between Phospho-SYT1 (Ser309) and SV2A in synaptic function?

The relationship between Phospho-SYT1 (Ser309) and SV2A represents an important area of investigation in synaptic biology:

Key interaction aspects:

• SV2A and synaptotagmin-1 are both integral membrane proteins of synaptic vesicles

• Research has shown that phosphorylation of SV2A at Thr84 controls its interaction with synaptotagmin-1

• This indicates a phosphorylation-dependent regulatory mechanism between these two proteins

• SV2A is implicated as a "phospho-switch" for synaptic vesicle recycling

Functional significance:

• The phosphorylation states of both proteins may coordinate aspects of vesicle priming, fusion, and recycling

• Their interaction might be reciprocally regulated by their respective phosphorylation states

• The temporal sequence of phosphorylation events could determine the progression of the synaptic vesicle cycle

Research approaches to investigate this relationship:

• Co-immunoprecipitation studies using phospho-specific antibodies

• Proximity ligation assays to visualize interactions in situ

• Functional studies using phosphomimetic or phospho-deficient mutants of both proteins

• Live imaging using fluorescently tagged constructs to monitor dynamic interactions

Understanding this phosphorylation-dependent relationship provides important insights into the molecular mechanisms governing synaptic transmission and plasticity.

What methodological approaches can be used to study dynamic phosphorylation of SYT1 at Ser309 in live neurons?

Studying the dynamic phosphorylation of SYT1 at Ser309 in living neurons requires specialized approaches:

Fluorescent biosensors:

• Development of FRET-based sensors that report on SYT1 phosphorylation state

• Constructs containing SYT1 flanked by appropriate fluorophores that change FRET efficiency upon phosphorylation

• Requires careful design to ensure phosphorylation-induced conformational changes affect FRET signal

Phosphorylation-sensitive fluorescent proteins:

• Adaptation of existing phosphorylation sensors to the SYT1 Ser309 site

• Integration of phospho-binding domains that change fluorescence properties upon binding to phosphorylated SYT1

SYT1-pHluorin with phospho-specific readouts:

• Building on existing SYT1-pHluorin constructs used in vesicle cycling studies

• Combining with other fluorescent indicators to correlate vesicle cycling with phosphorylation events

Optogenetic manipulation of kinases/phosphatases:

• Light-activated kinases targeted to synapses to induce SYT1 phosphorylation

• Temporal control of phosphorylation to study functional consequences

Correlative approaches:

• Live imaging followed by rapid fixation and phospho-specific immunostaining

• Allows correlation of functional synaptic events with subsequent phosphorylation analysis

Calcium imaging coupled with phosphorylation sensors:

• Dual monitoring of calcium dynamics and SYT1 phosphorylation

• Helps establish temporal relationship between calcium signals and phosphorylation events

These advanced techniques provide opportunities to understand the timing, localization, and functional consequences of SYT1 phosphorylation in intact neuronal circuits.

What are the key considerations when selecting a Phospho-SYT1 (Ser309) antibody for specific research applications?

Researchers should consider several factors when selecting an appropriate Phospho-SYT1 (Ser309) antibody:

Different products have specific optimization recommendations. For instance, dilution factors range from 1:100-1:400 for immunofluorescence and 1:1000 for Western blotting across different manufacturers .

How does phosphorylation at Ser309 compare with other phosphorylation sites on Synaptotagmin-1?

Synaptotagmin-1 contains multiple phosphorylation sites that serve different regulatory functions:

The various phosphorylation sites likely work in concert to fine-tune synaptotagmin-1 function through:

• Altering binding affinity for calcium ions

• Modifying interactions with membrane phospholipids

• Regulating protein-protein interactions with other synaptic components

• Potentially affecting the spatial positioning of synaptotagmin within the presynaptic terminal

Understanding the interplay between these different phosphorylation events requires sophisticated experimental approaches that can detect multiple modifications simultaneously.