SYT13 Antibody

What is SYT13 and why is it significant in research?

SYT13 (Synaptotagmin 13) is a member of the synaptotagmin family of proteins that function as membrane trafficking proteins. It has a calculated molecular weight of 46.9 kDa and is composed of canonical domains including an N-terminal transmembrane region, two C-terminal cytoplasmic C2-domains, and a connecting sequence between these regions .

Unlike typical synaptotagmins, SYT13 is an atypical member that lacks an extracellular N-terminus sequence and is evolutionarily conserved with high homology between human and rodent sequences . SYT13 is significant in research due to its diverse expression patterns and functions, including:

• Expression in brain, heart, lung, testis, spleen, kidney, pancreas, and intestinal tissues

• Involvement in vesicle docking to plasma membranes

• Critical roles in cellular morphogenesis and migration

• Protective functions in motor neurons in neurodegenerative conditions

• Implication in cancer metastasis and progression

What applications are SYT13 antibodies suitable for in experimental research?

SYT13 antibodies have been validated for multiple experimental applications, with varying protocols depending on the specific antibody and supplier. The primary applications include:

When designing experiments, researchers should consider that different antibodies may perform differently across applications, with some optimized for specific techniques .

How should I validate SYT13 antibody specificity for my research?

Validating antibody specificity is critical for ensuring reliable experimental results. For SYT13 antibodies, consider implementing these validation strategies:

• Positive and negative control tissues/cells:

  • Positive controls: Use tissues with known SYT13 expression such as brain, pancreas, or intestinal samples

  • Negative controls: Use knockout or knockdown models where available, or tissues with minimal expression

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide before application

  • Observe signal reduction/elimination in Western blot or immunostaining

  • Example: HuvEc cell extracts with and without immunizing peptide competition showed elimination of the 47 kDa band when peptide was present

• Molecular weight verification:

  • Confirm detection of bands at expected molecular weight (approximately 47 kDa)

  • Note that SYT13 may also be detected at 66 kDa in some samples, possibly representing post-translational modifications or splice variants

• Cross-validation with multiple antibodies:

  • Use antibodies recognizing different epitopes of SYT13

  • Compare staining patterns across antibodies targeting N-terminal vs. internal regions

What are the optimal fixation and retrieval methods for SYT13 immunohistochemistry?

Successful immunohistochemical detection of SYT13 requires appropriate sample preparation:

• Fixation:

  • Paraformaldehyde (PFA) fixation is suitable for immunofluorescence applications

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are compatible with many SYT13 antibodies

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) is recommended

  • Primary options:

    • TE buffer at pH 9.0 (preferred for many applications)

    • Citrate buffer at pH 6.0 (alternative method)

  • Optimal retrieval conditions may vary between antibodies and should be empirically determined

• Permeabilization:

  • For cell cultures and frozen sections, Triton X-100 permeabilization improves antibody penetration

  • For FFPE tissues, permeabilization is typically achieved during the antigen retrieval process

• Blocking:

  • Standard blocking with serum or BSA (typically 1-5%) reduces background

  • Include detergent (0.1-0.3% Triton X-100) in blocking solution for improved penetration

How can I differentiate between specific and non-specific SYT13 antibody binding?

Distinguishing specific from non-specific signals is crucial for accurate data interpretation:

• Expected SYT13 expression patterns:

  • In normal tissues: Strong expression in brain, variable expression in heart, lung, testis, spleen, kidney, pancreas

  • Subcellular localization: Associated with vesicles, plasma membrane, and potentially polarized domains in migrating cells

  • During development: Localized to the apical membrane of endocrine precursors and to the front domain of egressing endocrine cells in pancreatic development

• Potential sources of non-specific binding:

  • Cross-reactivity with other synaptotagmin family members (there are multiple SYT proteins with structural similarities)

  • Fc receptor binding in immune cells

  • Endogenous peroxidase or biotin activity

  • Hydrophobic interactions with fixatives

• Controls to implement:

  • Isotype controls matching the host species and antibody class

  • Secondary antibody-only controls

  • Blocked primary antibody (pre-incubated with immunizing peptide)

  • Progressive dilution series to identify optimal signal-to-noise ratio

What explains the multiple bands sometimes observed in Western blots with SYT13 antibodies?

When multiple bands appear in Western blots using SYT13 antibodies, consider these possible explanations:

• Expected band patterns:

  • Primary band at approximately 47 kDa (calculated molecular weight)

  • Additional band at approximately 66 kDa observed with some antibodies

• Biological explanations for multiple bands:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Alternative splice variants

  • Protein degradation products

  • Protein dimers or complexes resistant to denaturation

• Technical considerations:

  • Sample preparation conditions (reducing vs. non-reducing)

  • Gel percentage (affects resolution of proteins)

  • Transfer efficiency (particularly for higher molecular weight proteins)

  • Antibody specificity (some may detect related synaptotagmin family members)

• Validation approach:

  • Compare band patterns across multiple antibodies targeting different epitopes

  • Perform knockdown/knockout experiments to confirm specificity

  • Use appropriate positive control samples (e.g., HuvEc, NT2D1, U87-MG, or SK-N-SH cell lysates)

How does SYT13 function differ from other synaptotagmin family members in cellular processes?

Unlike canonical synaptotagmins that mediate calcium-dependent exocytosis, SYT13 exhibits several distinctive characteristics:

• Structural distinctions:

  • Lacks an extracellular N-terminus sequence present in typical synaptotagmins

  • Contains the canonical transmembrane region and C2-domains but may have different calcium-binding properties

• Functional distinctions:

  • Involved in vesicle trafficking rather than primarily synaptic transmission

  • Participates in cell morphogenesis and migration, particularly during development

  • Interacts with phosphatidylinositol phospholipids for polarized localization

  • Influences cell-matrix adhesion by internalizing a subset of plasma membrane proteins, including α6β4 integrins

• Tissue distribution differences:

  • Broader expression pattern compared to neuronal-specific synaptotagmins

  • Significant expression in non-neuronal tissues including pancreas, intestine, and cancer cells

• Experimental approaches for comparative studies:

  • Co-immunoprecipitation to identify distinct binding partners

  • Live-cell imaging with fluorescently tagged constructs to observe trafficking dynamics

  • Calcium dependence assays to determine functional differences in response to calcium

What role does SYT13 play in pancreatic endocrine development and how can this be experimentally investigated?

SYT13 has been identified as a critical factor in pancreatic endocrine cell development, with significant implications for diabetes research:

• Developmental functions:

  • Orchestrates pancreatic endocrine cell egression during islet formation

  • Localizes to the apical membrane of endocrine precursors and to the front domain of egressing endocrine cells

  • Facilitates the switch from apical-basal to front-rear polarization during endocrine cell egression

  • Modulates cell-matrix adhesion by internalizing plasma membrane proteins including α6β4 integrins

• Impact on cell fate determination:

  • Knockout of Syt13 impairs endocrine cell egression and skews the α-to-β-cell ratio

  • Deletion of Syt13 in endocrine precursors, but not in newly generated insulin-expressing cells, increases the α-to-β cell ratio

  • Acts downstream of Neurogenin 3 (Ngn3) in endocrine precursors

• Experimental approaches for investigation:

  • Lineage tracing using Syt13 reporter mouse models (e.g., NVF mouse models)

  • Conditional knockout models targeting specific developmental stages

  • Single-cell RNA sequencing to identify transcriptional networks

  • Time-lapse imaging of pancreatic explant cultures

  • FACS isolation of Ngn3+ cells followed by qPCR analysis to examine the effect of Syt13 on endocrine lineage markers

How is SYT13 implicated in cancer progression and what experimental models are appropriate for its study?

SYT13 has emerging roles in cancer biology that may represent potential therapeutic targets:

• Cancer-associated functions:

  • Upregulated in several cancer types including gastric cancer, colorectal cancer, and lung adenocarcinoma

  • Implicated in cancer cell metastasis and progression

  • Inhibition using antisense oligonucleotides hampers cancer metastasis and progression

• Experimental approaches for cancer studies:

  • Immunohistochemical analysis of human cancer tissues using validated SYT13 antibodies

  • Correlation of SYT13 expression with clinical outcomes and pathological features

  • siRNA or CRISPR-based knockdown/knockout in cancer cell lines

  • Antisense oligonucleotide inhibition strategies

  • Xenograft models to assess in vivo effects on tumor growth and metastasis

• Cell line models with documented SYT13 expression:

  • NT2D1, U87-MG, and SK-N-SH cell lines show detectable SYT13 expression by Western blot

  • HepG2 cells have been used as positive controls for Western blot detection

  • A549 cells have been used for immunofluorescent analysis

What is the protective role of SYT13 in motor neuron diseases and how can researchers investigate this function?

SYT13 has been identified as having neuroprotective properties relevant to motor neuron diseases:

• Neuroprotective functions:

  • Plays a protective function in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA)

  • Expression increases in several brain regions after contextual fear conditioning, suggesting roles in neural plasticity

• Experimental models for neurodegeneration studies:

  • ALS patient-derived motor neurons

  • SMA patient-derived motor neurons

  • Transgenic mouse models of ALS (SOD1, TDP-43, C9orf72)

  • Zebrafish models of motor neuron disease

• Research approaches:

  • Overexpression studies to assess neuroprotective effects

  • Analysis of SYT13 expression in post-mortem brain tissues from patients with neurodegenerative diseases

  • Evaluation of SYT13 as a biomarker for disease progression

  • Investigation of SYT13-mediated mechanisms of neuroprotection

  • Screening for small molecules that enhance SYT13 expression or function

What are the optimal conditions for using SYT13 antibodies in live-cell imaging experiments?

For researchers interested in dynamic processes involving SYT13, live-cell imaging presents unique challenges:

• Antibody format considerations:

  • Select non-conjugated primary antibodies that can be directly labeled with fluorophores

  • Consider Fab fragments for reduced steric hindrance and improved penetration

  • Antibodies targeting extracellular epitopes are preferred for non-permeabilized conditions

• Cell preparation:

  • Culture cells on imaging-compatible substrates (glass-bottom dishes, coverslips)

  • Minimize background by using phenol red-free, serum-reduced media during imaging

  • Consider physiological buffers that maintain cell viability during extended imaging sessions

• Alternative approaches:

  • CRISPR knock-in of fluorescent tags to endogenous SYT13

  • Transfection with fluorescently tagged SYT13 constructs (noting potential overexpression artifacts)

  • Use of fluorescently labeled ligands or interacting partners

• Validation strategies:

  • Compare live-cell patterns with fixed-cell immunostaining

  • Verify specificity through knockdown/knockout controls

  • Perform co-localization studies with known vesicle markers

How should researchers design experiments to investigate the interaction of SYT13 with phosphatidylinositol phospholipids?

The interaction between SYT13 and phosphatidylinositol phospholipids is critical for its polarized localization and function:

• Biochemical approaches:

  • Lipid overlay assays using purified SYT13 protein

  • Liposome binding assays with varying phospholipid compositions

  • Surface plasmon resonance to measure binding kinetics

  • Mutagenesis of putative lipid-binding domains in SYT13

• Cellular approaches:

  • Co-localization studies with phosphoinositide biosensors

  • Manipulation of phosphoinositide levels using phosphatase/kinase inhibitors

  • Expression of dominant-negative phosphoinositide-modifying enzymes

  • FRET-based assays to detect direct interactions

• Structural considerations:

  • The C2 domains of SYT13 likely mediate phospholipid interactions

  • Comparative analysis with other synaptotagmin family members may identify unique binding properties

  • Molecular modeling based on crystal structures of related proteins

• Functional readouts:

  • Effects on SYT13 localization and trafficking

  • Consequences for cell polarity and migration

  • Impact on vesicle docking and membrane fusion events