SYTL4 Antibody

What is SYTL4 and why is it relevant to cancer research?

SYTL4 (Synaptotagmin-like 4) is a Rab effector involved in vesicle transport that has recently gained attention in cancer research, particularly in triple-negative breast cancer (TNBC). Research has identified SYTL4 as a novel chemoresistant gene in TNBC, with high expression levels indicating poor prognosis specifically in taxane-treated TNBC patients. Mechanistically, SYTL4 downregulates microtubule stability and confers paclitaxel resistance by interacting with microtubules through its middle region containing the linker and C2A domain. This interaction enables SYTL4 to bind microtubules and inhibit in vitro microtubule polymerization, making it a potential therapeutic target for overcoming chemoresistance in cancer treatment.

How do I choose the appropriate SYTL4 antibody for my specific research application?

When selecting a SYTL4 antibody, consider these key factors: (1) Validated applications - verify the antibody has been tested for your application (Western blot, IHC, ICC/IF, IP); (2) Species reactivity - ensure compatibility with your experimental model (human, mouse, rat); (3) Antibody type - polyclonal antibodies like Proteintech's 12128-1-AP offer broader epitope recognition while monoclonal antibodies like Merck's clone 7D2.2 provide higher specificity; (4) Published validation - check for antibodies used in peer-reviewed publications; and (5) Immunogen information - antibodies raised against different regions of SYTL4 may perform differently. For example, Proteintech's polyclonal antibody (12128-1-AP) has been validated for WB, IHC, IF, and IP applications in human and mouse samples, with observed molecular weight at 76 kDa.

What are the alternative names for SYTL4 that might appear in literature?

SYTL4 is referenced by several alternative names in scientific literature that researchers should be aware of when conducting comprehensive literature searches. These aliases include: exophilin-2, granuphilin (or granuphilin-a), SLP4 (synaptotagmin-like protein 4), FLJ40960, and DKFZp451P0116. SYTL4 belongs to the synaptotagmin-like protein (Slp) family, characterized by an N-terminal Slp homology domain (SHD) that functions as a Rab27-binding domain. When searching databases or literature, including these alternative names will ensure you retrieve all relevant research on this protein. The NCBI Gene ID for SYTL4 is 94121, and its UniProt ID is Q96C24, which can further assist in database searches.

What are the recommended protocols for SYTL4 detection by Western blotting?

For optimal SYTL4 detection by Western blotting, follow these methodological guidelines: (1) Sample preparation - use RIPA or western/IP lysis buffer for protein extraction; (2) Loading amount - load 20-40 μg of total protein per lane; (3) Antibody dilution - for Proteintech's 12128-1-AP antibody, use a dilution range of 1:500-1:3000, while Merck's monoclonal antibody 7D2.2 works at 0.5 μg/mL; (4) Expected molecular weight - look for a band at approximately 76 kDa, which corresponds to the calculated molecular weight of 671 amino acids; (5) Positive controls - HeLa cells, U2OS cells, and mouse pancreas tissue have been validated for SYTL4 expression. For blocking and incubation, use 5% non-fat milk in TBST, and include appropriate loading controls. Remember that different antibodies may require optimization of these conditions for your specific experimental system.

How should I perform immunohistochemistry (IHC) to detect SYTL4 in tissue samples?

For successful SYTL4 immunohistochemistry, implement this methodological approach: (1) Fixation - use 10% neutral-buffered formalin for tissue fixation and paraffin embedding; (2) Antigen retrieval - perform heat-induced epitope retrieval using TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 as an alternative; (3) Blocking - block endogenous peroxidase activity with 3% H₂O₂ and use 5-10% normal serum for protein blocking; (4) Primary antibody - for Proteintech's antibody (12128-1-AP), use a dilution range of 1:50-1:500 and incubate overnight at 4°C; (5) Detection system - use a polymer-based detection system for higher sensitivity; (6) Counterstaining - use hematoxylin for nuclear counterstaining. SYTL4 staining is primarily cytoplasmic in tumor cells, and expression can be quantified using the H-score method (ranging from 0-12, with scores 0-7 considered low expression and 8-12 considered high expression). Human breast cancer tissue has been validated as a positive control for SYTL4 IHC.

What approaches are effective for studying SYTL4 knockdown in cellular models?

For effective SYTL4 knockdown studies, consider these methodological approaches: (1) siRNA transfection - use Lipofectamine RNAiMAX with 50 nM siRNA in antibiotic-free medium; validated target sequences include 5'-GCAGCATGATGAGCATCTA-3', 5'-GTCTGGTTGTCCATGTGAA-3', 5'-GCTGGCCTATGCTGATGAA-3', 5'-GGATATGGAAGAGGAAGAA-3', and 5'-GGATTTGATTCTCAGTGTT-3'; (2) Transfection efficiency - seed 3000 cells/well in 96-well plates and verify knockdown by Western blot 48-72 hours post-transfection; (3) Functional assays - measure microtubule stability through immunofluorescence of α-tubulin after SYTL4 knockdown; (4) Chemosensitivity testing - assess paclitaxel sensitivity changes using cell viability assays; (5) Control selection - always include a scrambled siRNA control to account for non-specific effects of the transfection process. For advanced applications, consider generating stable knockdown cell lines using shRNA or CRISPR-Cas9 systems for long-term studies of SYTL4 function in cancer cell models.

How can I investigate SYTL4-microtubule interactions in my research model?

To investigate SYTL4-microtubule interactions, implement these advanced methodological approaches: (1) Co-immunoprecipitation - use anti-SYTL4 antibodies to pull down protein complexes and probe for tubulin, or vice versa; (2) In vitro binding assays - perform His-tag pull-down assays with purified His-SYTL4 protein incubated with tubulins and Ni²⁺-NTA beads, then detect interactions via Western blot with anti-α-tubulin and anti-SYTL4 antibodies; (3) Immunofluorescence co-localization - perform dual staining with SYTL4 and α-tubulin antibodies, followed by confocal microscopy analysis using Pearson's correlation coefficient to quantify co-localization; (4) In vitro microtubule polymerization assays - assess how purified SYTL4 affects tubulin polymerization kinetics; (5) Domain mapping - create truncated SYTL4 constructs to identify which regions (particularly the middle region containing the linker and C2A domain) are crucial for microtubule binding. These methods will help elucidate the molecular mechanisms by which SYTL4 influences microtubule dynamics and stability in the context of chemoresistance.

What are common technical challenges when working with SYTL4 antibodies and how can they be resolved?

When working with SYTL4 antibodies, researchers commonly encounter these technical challenges and solutions: (1) Non-specific bands in Western blots - optimize blocking conditions (try 5% BSA instead of milk), increase washing stringency, and titrate antibody concentration (start with 1:1000 dilution for Proteintech's antibody); (2) Weak or no signal in IHC - enhance antigen retrieval by testing both TE buffer pH 9.0 and citrate buffer pH 6.0, extend primary antibody incubation time to overnight at 4°C, and use polymer-based detection systems for higher sensitivity; (3) High background in immunofluorescence - increase blocking time (2 hours at room temperature), use antibody diluent containing 0.1-0.3% Triton X-100 for better penetration, and ensure secondary antibody compatibility; (4) Cross-reactivity concerns - validate antibody specificity using SYTL4 knockdown controls; (5) Inconsistent results between experiments - standardize protocols, prepare fresh working solutions, and monitor protein degradation by adding protease inhibitors to lysates. Always include positive controls (HeLa cells, U2OS cells, or mouse pancreas tissue) when optimizing new SYTL4 antibody applications.

How can I validate the specificity of my SYTL4 antibody to ensure reliable experimental results?

To rigorously validate SYTL4 antibody specificity, implement these critical validation methods: (1) Knockdown/knockout controls - transfect cells with SYTL4-specific siRNAs (using sequences like 5'-GCAGCATGATGAGCATCTA-3') or generate CRISPR knockout cells and confirm reduced signal in Western blot or immunostaining; (2) Overexpression controls - transfect cells with SYTL4 expression vectors and verify increased signal; (3) Pre-absorption tests - pre-incubate antibody with purified SYTL4 antigen before application to samples and confirm signal reduction; (4) Multi-antibody approach - compare results using antibodies targeting different SYTL4 epitopes; (5) Mass spectrometry verification - perform immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein. For additional validation, check if the antibody recognizes the expected molecular weight (76 kDa) and expected subcellular localization (primarily cytoplasmic). Antibodies like Proteintech's 12128-1-AP have been validated through knockdown experiments as cited in multiple publications, providing greater reliability for research applications.

How might SYTL4 function as a potential therapeutic target in cancer treatment strategies?

SYTL4 shows promising potential as a therapeutic target in cancer treatment, particularly for overcoming taxane resistance in triple-negative breast cancer. To explore this direction: (1) Target validation - confirm SYTL4's causal role in chemoresistance through gene editing approaches and patient-derived xenograft models; (2) Drug discovery approaches - develop small molecule inhibitors targeting the SYTL4-microtubule interaction, focusing on the middle region containing the linker and C2A domain which mediates microtubule binding; (3) Combination therapy strategies - test SYTL4 inhibition in combination with standard-of-care taxanes to restore chemosensitivity; (4) Biomarker development - establish SYTL4 expression as a predictive biomarker for taxane response using validated immunohistochemistry protocols with clear cutoff values (H-scores 0-7 vs. 8-12); (5) Delivery strategies - explore nanoparticle-based or antibody-drug conjugate approaches to specifically target cancer cells with high SYTL4 expression. This research direction could lead to novel therapeutic strategies for patients with chemoresistant tumors characterized by high SYTL4 expression.

What is currently known about the regulation of SYTL4 expression in different cellular contexts?

The regulation of SYTL4 expression across different cellular contexts remains an emerging area of research with several key insights: (1) Tissue distribution - SYTL4 shows variable expression patterns across tissues, with notable expression in pancreatic tissue (as evidenced by positive Western blot detection in mouse pancreas) and certain cancer cell lines like HeLa and U2OS; (2) Cancer-specific upregulation - SYTL4 is upregulated in certain cancer types, particularly in triple-negative breast cancer, where its expression correlates with poor prognosis in taxane-treated patients; (3) Transcriptional regulation - while specific transcription factors controlling SYTL4 expression are not fully characterized in the provided search results, investigating promoter analysis and transcription factor binding sites would be valuable future research directions; (4) Post-transcriptional regulation - examining potential microRNA regulation of SYTL4 mRNA stability and translation efficiency could provide additional insights into its expression control; (5) Protein stability - research into the post-translational modifications and degradation pathways affecting SYTL4 protein levels would further elucidate its regulation. Understanding these regulatory mechanisms could identify additional intervention points for modulating SYTL4 activity in disease contexts.

What methodological approaches can be used to study SYTL4's role in vesicle transport beyond its microtubule interactions?

To investigate SYTL4's role in vesicle transport beyond microtubule interactions, implement these specialized methodological approaches: (1) Rab27 interaction studies - analyze SYTL4-Rab27 binding through co-immunoprecipitation and proximity ligation assays, as SYTL4 contains an N-terminal Slp homology domain (SHD) that functions as a Rab27-binding domain; (2) Live-cell imaging - use fluorescently-tagged SYTL4 combined with vesicle markers to track vesicle dynamics and trafficking patterns in real-time; (3) Super-resolution microscopy - employ techniques like STORM or PALM to visualize SYTL4-positive vesicles with nanometer precision; (4) Cargo identification - perform proteomic analysis of SYTL4-associated vesicles to identify transported molecules; (5) Secretion assays - measure the impact of SYTL4 manipulation on exocytosis rates of specific cargo molecules. These approaches will help elucidate how SYTL4 functions in the broader context of membrane trafficking pathways, potentially revealing additional mechanisms by which it influences cellular processes beyond its known effects on microtubule stability and chemoresistance.

How do commercially available SYTL4 antibodies compare in terms of specificity, applications, and species reactivity?

A comprehensive comparison of commercially available SYTL4 antibodies reveals important differences that can guide selection for specific research applications:

When selecting between these antibodies, consider that polyclonal antibodies (Proteintech, Novus) typically offer broader epitope recognition but potential batch-to-batch variability, while monoclonal antibodies (Merck) provide higher specificity and consistency. Proteintech's antibody shows the broadest validated application range and species reactivity, making it versatile for multiple experimental approaches. For highly specific Western blot applications in human samples, the Merck monoclonal antibody may provide advantages. Always validate any selected antibody in your specific experimental system before proceeding with critical experiments.

What emerging technologies might enhance our understanding of SYTL4 function in normal physiology and disease?

Emerging technologies poised to advance our understanding of SYTL4 function include: (1) Single-cell proteomics - to reveal cell-type specific expression patterns and protein interactions of SYTL4 within heterogeneous tissues; (2) CRISPR-based screens - using focused libraries targeting vesicle trafficking and microtubule-related genes to identify synthetic lethal interactions with SYTL4; (3) Cryo-electron microscopy - to solve the structure of SYTL4-microtubule complexes at near-atomic resolution, revealing precise binding interfaces; (4) Optogenetic approaches - developing light-activatable SYTL4 variants to control its function with spatiotemporal precision in living cells; (5) Patient-derived cerebral organoids - to study SYTL4's potential roles in neurological contexts beyond its established functions in cancer and vesicle transport. These technological approaches could reveal unexpected functions of SYTL4 and provide deeper mechanistic insights into how it contributes to both normal cellular processes and pathological conditions, potentially expanding therapeutic applications beyond cancer treatment.

How can understanding SYTL4's role in microtubule dynamics inform the development of novel chemotherapeutic strategies?

Understanding SYTL4's role in microtubule dynamics opens several promising avenues for novel chemotherapeutic strategies: (1) Combination therapy approaches - develop protocols combining taxanes with SYTL4 inhibitors to overcome resistance, potentially allowing for lower effective doses of chemotherapy; (2) Patient stratification - implement SYTL4 expression testing via immunohistochemistry to identify patients likely to benefit from alternative treatment strategies when high expression (H-scores 8-12) is detected; (3) Drug delivery innovations - design microtubule-targeting nanoparticles that simultaneously inhibit SYTL4 function to enhance drug efficacy; (4) Structural biology-guided drug design - use information about SYTL4's interaction with microtubules through its middle region containing the linker and C2A domain to design specific inhibitors of this interaction; (5) Synthetic lethality approaches - identify genes that, when inhibited alongside SYTL4 modulation, cause catastrophic failure of microtubule networks in cancer cells while sparing normal cells. These strategies could significantly advance personalized medicine approaches for patients with chemoresistant cancers, particularly those with high SYTL4 expression that confers paclitaxel resistance through microtubule destabilization.