SYNGR1 Antibody

What is SYNGR1 and what cellular functions does it regulate?

Synaptogyrin 1 (SYNGR1) is one of the most abundant vesicle components in neuronal cells, playing critical roles in the regulation of neurotransmitter release and synaptic plasticity. It belongs to a family of tyrosine-phosphorylated membrane proteins with three primary isoforms: two neuronal forms (synaptogyrin-1 and -3) and one ubiquitous form (synaptogyrin-2). SYNGR1 functions specifically as a regulator of Ca²⁺-dependent exocytosis in synaptic vesicles, making it essential for normal neuronal communication . The protein's strategic localization and functional properties highlight its importance in maintaining synaptic integrity and function. Understanding these basic characteristics is fundamental before designing experiments targeting this protein.

What are the key specifications researchers should know about commercially available SYNGR1 antibodies?

When selecting SYNGR1 antibodies for experimental applications, researchers should consider several critical specifications:

• Antibody formats: Available as both polyclonal (rabbit) and recombinant antibodies, with different performance characteristics depending on experimental needs .

• Molecular weight considerations: The calculated molecular weight of SYNGR1 is approximately 25 kDa, but the observed molecular weight in experimental conditions typically ranges between 28-30 kDa, which is important for proper identification in Western blot applications .

• Species reactivity: Most commercially available antibodies show validated reactivity with human, mouse, rat, and pig samples, allowing for comparative studies across species .

• Applications compatibility: SYNGR1 antibodies are validated for multiple techniques including Western Blot (WB), Immunofluorescence (IF/ICC), Immunohistochemistry (IHC), and ELISA, though optimization is required for each specific application .

How should researchers approach experimental design when first working with SYNGR1 antibodies?

When initiating research with SYNGR1 antibodies, a methodical approach to experimental design is essential. First, researchers should perform antibody validation using positive controls such as brain tissue samples (rat, mouse, or pig brain) where SYNGR1 expression is well-documented . For cellular studies, A431 cells have been validated as appropriate positive controls for immunofluorescence applications .

Beginning with recommended dilutions is critical: for Western blot applications, start with 1:500-1:1000 for polyclonal antibodies or 1:5000-1:50000 for recombinant antibodies, titrating as needed for optimal signal-to-noise ratio . For immunofluorescence, initial dilutions of 1:50-1:500 are recommended, with subsequent optimization based on signal intensity and background levels .

Additionally, researchers should establish appropriate negative controls, such as secondary antibody-only controls and/or tissues known not to express SYNGR1, to confirm specificity. When analyzing results, be aware that the observed molecular weight may differ from calculated values due to post-translational modifications, which is normal for SYNGR1 detection .

What are the optimal conditions for maximizing specificity in SYNGR1 Western blot applications?

Achieving high specificity in SYNGR1 Western blot applications requires careful optimization of multiple parameters. For protein extraction, brain tissues should be homogenized in RIPA buffer supplemented with protease inhibitors to prevent degradation of the target protein. Membrane proteins like SYNGR1 benefit from sample preparation protocols that include mild detergents to maintain protein integrity .

For electrophoresis, 10-12% polyacrylamide gels provide optimal resolution for the 25-30 kDa size range where SYNGR1 is detected. Transfer efficiency can be enhanced using a wet transfer system with methanol-containing buffer at controlled temperature (4°C). When performing immunodetection, membrane blocking with 5% non-fat milk or BSA for 1-2 hours significantly reduces background signal .

Working with recombinant SYNGR1 antibodies (such as 84161-3-RR) often provides higher specificity and can be used at higher dilutions (1:5000-1:50000) compared to polyclonal alternatives, potentially resulting in cleaner blots with less background . For challenging samples, overnight primary antibody incubation at 4°C followed by extensive washing (minimum of 3-4 washes, 10 minutes each) can dramatically improve specificity while maintaining sensitivity .

How can researchers optimize SYNGR1 immunofluorescence protocols for different neural tissue types?

Optimizing SYNGR1 immunofluorescence protocols requires tissue-specific adaptations to address the unique challenges presented by different neural samples. For fixed tissues, antigen retrieval methods significantly impact SYNGR1 detection - heat-mediated retrieval using citrate buffer (pH 6.0) has proven effective for most brain tissue preparations . When working with brain sections, reducing autofluorescence can be achieved by treating samples with 0.1% sodium borohydride prior to blocking and primary antibody incubation .

For cultured neurons, fixation with 4% paraformaldehyde for 10-15 minutes at room temperature preserves SYNGR1 morphology while maintaining antibody accessibility to the target. Permeabilization should be performed gently, typically using 0.1% Triton X-100 for 5-10 minutes . Blocking solutions containing normal serum (5-10%) from the same species as the secondary antibody significantly reduces non-specific binding.

When examining subcellular localization, confocal microscopy with z-stack acquisition is recommended to accurately resolve SYNGR1's distribution within synaptic structures. For colocalization studies with other synaptic markers, sequential rather than simultaneous antibody incubations often yield better results, minimizing potential cross-reactivity between detection systems .

What experimental approaches can validate functional effects of SYNGR1 in Ca²⁺-dependent exocytosis?

Investigating SYNGR1's role in Ca²⁺-dependent exocytosis requires sophisticated experimental approaches that extend beyond simple protein detection. Researchers can implement calcium imaging combined with SYNGR1 immunolabeling to correlate calcium dynamics with SYNGR1 localization during stimulation. For functional studies, synaptic vesicle recycling assays using pH-sensitive fluorescent proteins (pHluorins) fused to synaptic vesicle proteins can monitor exocytosis in real-time while manipulating SYNGR1 expression .

Knockdown or knockout approaches using RNA interference or CRISPR-Cas9 technology, respectively, can establish causality between SYNGR1 levels and exocytosis efficiency. These genetic manipulations should be validated using the aforementioned SYNGR1 antibodies to confirm protein reduction or elimination . Following genetic manipulation, measuring neurotransmitter release via electrophysiological recordings provides direct functional readouts of SYNGR1's impact on synaptic transmission.

For mechanistic studies, co-immunoprecipitation experiments using SYNGR1 antibodies can identify protein interaction partners involved in the Ca²⁺-dependent machinery. Subsequent proximity ligation assays can confirm these interactions in intact cells, providing spatial context to the biochemical findings . These methodological approaches collectively provide a comprehensive assessment of SYNGR1's functional contributions to Ca²⁺-dependent exocytosis.

Why does the observed molecular weight of SYNGR1 differ from calculated predictions?

The discrepancy between SYNGR1's calculated molecular weight (25 kDa) and its observed molecular weight in experimental conditions (28-30 kDa) represents a common challenge in protein research that requires careful interpretation . This molecular weight shift primarily stems from post-translational modifications that alter protein mobility during electrophoresis. SYNGR1, as a synaptic vesicle protein, undergoes several modifications including glycosylation and phosphorylation that increase its apparent molecular weight .

Specifically, SYNGR1 contains multiple tyrosine phosphorylation sites, consistent with its classification as a tyrosine-phosphorylated protein. Additionally, as a membrane protein with transmembrane domains, SYNGR1 may not fully denature in standard SDS-PAGE conditions, further affecting its migration pattern . Sample preparation methods can also influence the observed molecular weight - incomplete denaturation or residual membrane associations can result in anomalous migration.

To address this issue when analyzing Western blot results, researchers should always run appropriate positive controls (such as brain tissue lysates) alongside experimental samples to establish the correct band identification . If necessary, phosphatase treatment of samples prior to electrophoresis can help confirm whether phosphorylation contributes to the observed weight difference.

How can researchers troubleshoot weak or absent SYNGR1 signal in immunofluorescence applications?

When confronting weak or absent SYNGR1 signal in immunofluorescence applications, systematic troubleshooting is essential to identify and resolve the underlying issues. First, confirm antibody viability by performing a straightforward Western blot on known positive control samples (brain tissue) to verify that the antibody still recognizes the target protein . Next, optimize fixation protocols as overfixation can mask epitopes - try reducing paraformaldehyde concentration (2-3% instead of 4%) or fixation duration.

Antigen retrieval methods may significantly improve signal strength by recovering epitopes lost during fixation. For SYNGR1 detection, heat-mediated antigen retrieval using citrate buffer (pH 6.0) has proven effective . Increasing antibody concentration beyond recommended ranges (e.g., using 1:50 dilution if 1:500 produces weak signal) can improve detection, though this should be accompanied by appropriate negative controls to monitor background increases.

Permeabilization efficiency should be assessed, particularly when working with membrane proteins like SYNGR1. Increasing Triton X-100 concentration (up to 0.3%) or extending permeabilization time may improve antibody access to target epitopes . For particularly challenging samples, signal amplification systems such as tyramide signal amplification can enhance detection sensitivity while maintaining specificity. Finally, microscope settings should be optimized - longer exposure times, increased gain, or using more sensitive detection systems may reveal signals previously below detection threshold .

What strategies can address experimental inconsistencies when studying SYNGR1 across different model systems?

Addressing experimental inconsistencies when studying SYNGR1 across different model systems requires systematic standardization and validation approaches. First, researchers should establish species-specific baseline expression profiles using validated antibodies that demonstrate cross-reactivity with the species being studied (human, mouse, rat, or pig) . This baseline characterization should include both qualitative (localization patterns) and quantitative (expression levels) assessments.

When comparing across models, standardize protein extraction methods to ensure equivalent cellular fractions are being analyzed. For membrane proteins like SYNGR1, differences in extraction efficiency can significantly impact results . Implementation of loading controls specific to the cellular compartment where SYNGR1 resides (synaptic vesicle markers) rather than general housekeeping proteins provides more accurate normalization for quantitative comparisons.

To address antibody-related variability, researchers should validate multiple antibodies against the same target in each model system to identify potential epitope-specific differences that might affect detection . For functional studies, complementary approaches such as mRNA expression analysis alongside protein detection can help reconcile discrepancies. When inconsistencies persist, researchers should consider species-specific post-translational modifications or splicing variants that might affect antibody recognition or protein function .

How is SYNGR1 implicated in the pathophysiology of schizophrenia?

SYNGR1 has emerged as a significant gene of interest in schizophrenia research, with multiple lines of evidence supporting its potential role in disease pathophysiology. Genetic studies have identified SYNGR1 as a positional candidate gene for schizophrenia, with specific mutations associated with disease risk . The functional significance of these mutations likely relates to SYNGR1's critical role in regulating Ca²⁺-dependent exocytosis and neurotransmitter release, processes frequently dysregulated in schizophrenia .

At the molecular level, SYNGR1 mutations may disrupt normal synaptic vesicle cycling, leading to altered neurotransmitter release kinetics and synaptic dysfunction. This dysfunction potentially contributes to the imbalances in dopaminergic and glutamatergic signaling that characterize schizophrenia pathophysiology . Research investigating SYNGR1 protein expression in post-mortem brain samples from schizophrenia patients has revealed altered expression patterns compared to control subjects.

For researchers investigating SYNGR1 in schizophrenia models, antibody-based approaches can quantify expression changes and alterations in subcellular localization. When designing such studies, careful selection of brain regions known to be affected in schizophrenia (prefrontal cortex, hippocampus) should be prioritized . Additionally, correlating SYNGR1 expression with other established schizophrenia-related markers can provide context for interpreting findings within the broader disease landscape.

What methodological approaches can assess SYNGR1's role in rheumatoid arthritis research?

Recent research has identified potential associations between SYNGR1 genetic variants and rheumatoid arthritis (RA), opening new avenues for investigating this protein beyond its neuronal functions . The single nucleotide polymorphism rs1893592 in the SYNGR1 gene has shown significant association with RA risk, with a combined odds ratio of 0.6791 (P-value = 4.50 × 10⁻⁹), suggesting a protective effect of the minor allele .

For researchers exploring this connection, several methodological approaches are recommended. Genotyping studies should include the rs1893592 SNP alongside other validated RA-associated variants to establish comprehensive genetic profiles. Patient cohorts should be carefully characterized regarding clinical parameters including rheumatoid factor (RF) seropositivity, anti-CCP antibody status, and disease activity measures like DAS28 scores .

To investigate the functional consequences of SYNGR1 variants, researchers can employ siRNA knockdown or CRISPR-Cas9 editing in relevant cell types (e.g., fibroblast-like synoviocytes from RA patients) followed by assessment of inflammatory cytokine production and signaling pathway activation . Immunophenotyping studies utilizing SYNGR1 antibodies can compare protein expression in peripheral blood mononuclear cells or synovial tissue from RA patients versus healthy controls, correlating expression with genetic variants and disease severity measures .

How can researchers effectively design studies to investigate SYNGR1 mutations associated with neurological disorders?

Designing effective studies to investigate SYNGR1 mutations associated with neurological disorders requires thoughtful integration of genetic, molecular, and functional approaches. Initially, researchers should conduct comprehensive mutation screening through targeted sequencing of SYNGR1 coding regions in patient populations with specific neurological phenotypes (schizophrenia, epilepsy, neurodevelopmental disorders) . This genetic analysis should include examination of both rare variants with potentially large effects and common polymorphisms with more modest contributions to disease risk.

For functional characterization of identified mutations, researchers can develop cellular models expressing mutant SYNGR1 variants using site-directed mutagenesis followed by transfection into neuronal cell lines or primary neurons . SYNGR1 antibodies are then essential for validating expression of the mutant proteins and assessing potential differences in subcellular localization compared to wild-type SYNGR1. Co-immunoprecipitation experiments can determine whether mutations disrupt normal protein-protein interactions critical for SYNGR1 function.

Advanced functional assays should focus on synaptic vesicle dynamics and Ca²⁺-dependent exocytosis, as these represent SYNGR1's primary neuronal functions . Techniques such as total internal reflection fluorescence (TIRF) microscopy combined with pH-sensitive vesicle markers can provide high-resolution analysis of how SYNGR1 mutations affect vesicle docking, fusion, and recycling. Electrophysiological recordings in neuronal cultures expressing mutant versus wild-type SYNGR1 can directly assess functional consequences on synaptic transmission .

How should researchers validate antibody specificity when working with SYNGR1?

Validating antibody specificity is essential for generating reliable and reproducible data in SYNGR1 research. A comprehensive validation strategy should include multiple complementary approaches. First, perform Western blot analysis using positive control tissues known to express SYNGR1 (brain tissues from rat, mouse, or pig) alongside negative control tissues where expression is expected to be absent or minimal . The detection of a single band at the expected molecular weight range (28-30 kDa) in positive controls provides initial evidence of specificity.

Genetic approaches provide definitive validation - testing the antibody in samples where SYNGR1 has been knocked down (siRNA) or knocked out (CRISPR-Cas9) should result in corresponding reduction or elimination of signal in both Western blot and immunofluorescence applications . For immunostaining applications, peptide competition assays where the antibody is pre-incubated with the immunizing peptide should abolish specific signal, confirming that binding occurs through the intended epitope.

Cross-validation using multiple antibodies targeting different epitopes of SYNGR1 increases confidence in specificity - concordant staining patterns across different antibodies strongly support true target recognition . When available, testing in cell lines engineered to overexpress SYNGR1 compared to control lines provides additional validation. Finally, correlation between protein detection (using antibodies) and mRNA expression (using qPCR or RNA-seq) across tissues or experimental conditions can further confirm specificity .

What quantitative approaches are most appropriate for analyzing SYNGR1 expression in comparative studies?

For immunofluorescence quantification, confocal microscopy with standardized acquisition parameters (laser power, detector gain, pinhole size) across all samples is essential for valid comparisons . Automated image analysis using specialized software can reduce bias and increase throughput when quantifying parameters such as signal intensity, puncta density, or colocalization with other markers. When analyzing tissue sections, standardized sampling approaches should be employed to ensure representative measurements from anatomically comparable regions.

Statistical analysis should account for biological and technical variability - using multiple biological replicates (different animals/patients) and technical replicates (multiple sections/blots per sample) . Non-parametric statistical tests are often appropriate when sample sizes are small or data doesn't follow normal distribution. For complex experiments comparing multiple groups or conditions, appropriate corrections for multiple comparisons should be applied to prevent false positive findings .

How can researchers reconcile conflicting findings regarding SYNGR1 expression or function in different experimental systems?

Reconciling conflicting findings regarding SYNGR1 expression or function requires systematic analysis of potential sources of variation across experimental systems. Begin by conducting a detailed comparison of methodological differences between studies - variations in antibody clones, detection systems, tissue preparation protocols, or quantification methods can significantly impact results . Document these methodological differences in a comprehensive comparison table to identify potential explanations for discrepancies.

Species differences represent another major source of variation - while SYNGR1 is highly conserved, subtle differences in protein sequence or post-translational modifications across species may affect antibody recognition or protein function . Similarly, developmental stage can significantly influence SYNGR1 expression patterns, so age-matching experimental subjects across studies is critical for valid comparisons.

When conflicting findings persist despite accounting for methodological and biological variables, consider designing bridging experiments that systematically test specific hypotheses about the source of discrepancies . This might involve directly comparing multiple antibodies within the same experimental system or replicating key experiments using standardized protocols across different model systems. Collaborative approaches where laboratories exchange materials or perform cross-validation studies can be particularly valuable for resolving persistent conflicts .