SYNGAP1 Antibody, Biotin conjugated

What is SYNGAP1 and why is it important in neuroscience research?

SYNGAP1 (Synaptic Ras GTPase activating protein 1) is a major signaling protein that plays a pivotal role in regulating fundamental molecular changes in dendritic spine synaptic morphological and functional modifications. It functions as a downstream component of NMDA receptor-associated signaling and of AMPA receptor trafficking to excitatory postsynaptic membrane. SYNGAP1 is mainly expressed in the hippocampus and cortex, making it an important target for researchers studying synaptic plasticity, neurodevelopment, and neurodevelopmental disorders .

SYNGAP1 is a member of the NMDAR signaling complex in excitatory synapses and may play a role in NMDAR-dependent control of AMPAR potentiation, AMPAR membrane trafficking, and synaptic plasticity . Recent research has revealed its expression in the murine suprachiasmatic nucleus, expanding our understanding of its distribution in the brain .

What are the structural and functional characteristics of SYNGAP1?

SYNGAP1 is a cytosolic protein without a signal peptide or transmembrane domains. The N-terminal half contains a Ras-GAP domain, along with regions homologous to Pleckstrin homology and a C2 domain potentially involved in binding Ca²⁺ and phospholipids. The alignment of the GAP domain with other Ras-GAPs suggests that amino acids in this domain are crucial for interacting with Ras and stimulating Ras-GTPase activity .

The C-terminal half consists of a repeat of 10 histidines that may be involved in metal chelation, several potential serine and tyrosine phosphorylation sites, and a T/SXV motif necessary for interaction with SAP102 . The calculated molecular weight of SYNGAP1 is 148 kDa (1343 amino acids) .

What are the key specifications of biotin-conjugated SYNGAP1 antibody?

The biotin-conjugated SYNGAP1 antibody (CSB-PA857006LD01HU) has the following specifications:

This antibody is intended for research use only and not for diagnostic or therapeutic procedures .

What are the recommended applications and dilutions for SYNGAP1 antibodies?

While specific dilution recommendations for the biotin-conjugated SYNGAP1 antibody may vary by lot and application, general guidance for SYNGAP1 antibodies includes:

It is strongly recommended to optimize the dilution for each specific application and sample type to obtain optimal results, as the optimal working dilution can be sample-dependent .

How should I design controls for experiments using biotin-conjugated SYNGAP1 antibody?

Proper controls are essential for ensuring the validity of results when using biotin-conjugated SYNGAP1 antibody:

• Positive Controls:

  • Use brain tissue samples (particularly from hippocampus or cortex) where SYNGAP1 is known to be expressed

  • Include mouse, rat, or pig brain tissues for cross-species validation

  • U-251 cells have been validated for IF/ICC applications

• Negative Controls:

  • Omit the primary antibody in parallel samples

  • Use tissues or cells known not to express SYNGAP1

  • For peptide competition assays, preincubate the antibody with the immunizing peptide (as demonstrated in Western blot analysis with SynGAP Blocking Peptide)

• Method-specific Controls:

  • For IHC/IF: Include autofluorescence or secondary-only controls

  • For Western blot: Use molecular weight markers to confirm the expected 148 kDa band

  • For co-localization studies: Include controls for each fluorophore channel

• Biological Controls:

  • Consider using SYNGAP1 knockout or knockdown models as specificity controls

  • Compare wildtype vs. mutant samples when studying disease-associated variants

What are the key considerations for optimizing western blot protocols with SYNGAP1 antibody?

Optimizing western blot protocols for SYNGAP1 detection requires attention to several factors:

• Sample Preparation:

  • Use fresh tissue when possible, particularly brain tissues where SYNGAP1 is highly expressed

  • For SCN tissue analysis, lyse in 100 μl of radioimmunoprecipitation assay buffer

  • For cortical tissue, use 200 μl of buffer

  • Load approximately 10 μg protein per lane

• Gel Selection and Transfer:

  • Use 12% SDS-PAGE gels for optimal separation of the 148 kDa SYNGAP1 protein

  • Transfer onto polyvinylidene difluoride membranes (e.g., Immobilon-P, EMD Millipore)

• Blocking and Antibody Incubation:

  • Block membrane for 1 hour at room temperature in 10% milk in PBST

  • Incubate with primary antibody (1:1000 dilution in 5% normal goat serum in PBST) overnight at 4°C

  • For biotin-conjugated antibodies, consider using streptavidin-HRP detection systems for enhanced sensitivity

• Detection and Visualization:

  • Use appropriate secondary detection reagents compatible with biotin conjugation

  • Consider enhanced chemiluminescence for sensitive detection

  • Expect to observe bands at approximately 148 kDa

How can SYNGAP1 antibodies be used to investigate synaptic protein complexes?

SYNGAP1 antibodies have proven valuable for investigating synaptic protein complexes:

• Protein Complex Immunoprecipitation:

  • SYNGAP1 antibodies can be used to pull down associated proteins in the NMDAR signaling complex

  • Research has revealed that 94% of Syngap1-interacting proteins identified by HiUGE-iBioID were also found in the Anks1b interactome, indicating they likely reside within the same supramolecular complex

  • Markov cluster analysis of their overlapping interactome identified a large PPI community (32 of 92 proteins) that is significantly enriched for pathways involved in "autistic disorder" and "glutamate receptor signaling"

• Mapping Protein Interactions:

  • Immunoprecipitation has confirmed regions essential for Syngap1-Anks1b interactions in vivo

  • Studies have demonstrated that remodeling of synaptic protein complexes may be a mechanism associated with synaptopathy in Syngap1 loss-of-function mutations

• Temporal Expression Analysis:

  • SYNGAP1 antibodies enable analysis of protein expression during different circadian timepoints

  • Studies examining SYNGAP1 expression at CT6 and CT14 have provided insights into temporal regulation

• Biotinylated RNA Pull-down Assays:

  • Biotin-conjugated SYNGAP1 antibodies facilitate the assessment of interactions between SYNGAP1 mRNA 3'UTR and endogenous RNA-binding proteins

What approaches can be used to study SYNGAP1 phosphorylation states?

Studying SYNGAP1 phosphorylation states is critical for understanding its regulation and function:

• Phospho-specific Antibodies:

  • Use antibodies targeting specific phosphorylation sites, such as anti-serine 1138 phosphorylated SYNGAP1 (SynGAP pSer1138)

  • Combine with total SYNGAP1 antibodies to determine the ratio of phosphorylated to total protein

• Immunohistochemical Analysis:

  • For tissue sections, process using DAB signal visualization with nickel intensification

  • When using phospho-specific antibodies, careful sample preparation is essential to preserve phosphorylation states

• Western Blot Analysis:

  • Run parallel blots probing for both total and phosphorylated SYNGAP1

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation states

• Activity-dependent Phosphorylation:

  • Design experiments to monitor changes in SYNGAP1 phosphorylation in response to neuronal activity

  • Consider co-staining with pERK antibodies to examine relationships between SYNGAP1 and ERK signaling pathways

How can researchers investigate SYNGAP1 mutations and their functional consequences?

SYNGAP1 mutations are associated with neurodevelopmental disorders, and their investigation requires specialized approaches:

• Protein-Protein Interaction Studies:

  • Use SYNGAP1 antibodies to compare interactions between wild-type and mutant proteins

  • Research has shown that ASD-associated SYNGAP1 mutations can disrupt binding with AnkS1b

• Electrophysiological Analysis:

  • Combine antibody-based protein detection with electrophysiological methods like multielectrode array (MEA) systems

  • This approach allows monitoring of electrophysiological activities longitudinally during neurodevelopment

  • Studies have observed elevated firing rates and burst activities in Syngap1-gRNA treated neurons at specific developmental time points (DIV11), which phenocopies previous findings of Syngap1 LOF accelerating network activity specifically during synaptogenesis periods

• Molecular Consequence Analysis:

  • Investigate how mutations affect SYNGAP1 splicing using biotinylated RNA pull-down assays

  • Research has identified UTR variants in ALS patients that cause aberrant SYNGAP1 splicing

• Genetic Interaction Studies:

  • Examine how SYNGAP1 mutations interact with other genes

  • Studies have shown that further depletion of Anks1b exacerbates the abnormal heightened neural activity linked to Syngap1 mutation, primarily during the period of synaptogenesis (DIV 11)

What are common challenges when using SYNGAP1 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with SYNGAP1 antibodies:

• High Molecular Weight Protein Detection:

  • Challenge: SYNGAP1 is a large protein (148 kDa) that can be difficult to transfer efficiently

  • Solution: Optimize transfer conditions using lower voltage for longer times or specialized transfer systems for high molecular weight proteins

• Tissue-Specific Expression:

  • Challenge: Expression levels vary significantly between brain regions

  • Solution: Select appropriate positive controls based on known expression patterns (hippocampus and cortex show highest expression)

  • For specific regions like the suprachiasmatic nucleus, pool tissue from multiple animals (e.g., 3 animals) to create sufficient sample material

• Background in Immunohistochemistry:

  • Challenge: High background staining in brain tissues

  • Solution:

    • Optimize blocking conditions (10% normal goat serum in 1X PBST for 1 hour at room temperature)

    • Include appropriate tissue processing steps (e.g., incubation in 0.3% hydrogen peroxide in PBST for 20 minutes at room temperature)

    • For biotin-conjugated antibodies, consider using avidin/biotin blocking kits to reduce endogenous biotin signals

• Antibody Specificity:

  • Challenge: Cross-reactivity with other proteins

  • Solution: Validate specificity using peptide competition assays with the specific immunizing peptide

How should researchers interpret differences in SYNGAP1 expression patterns across brain regions?

Interpreting SYNGAP1 expression patterns requires careful consideration of several factors:

• Regional Variation Analysis:

  • SYNGAP1 is predominantly expressed in hippocampus and cortex, but also detected in other regions like the suprachiasmatic nucleus

  • Immunohistochemical staining has revealed SynGAP staining in mouse globus pallidus that is markedly stronger than in the striatum

  • In rat brain sections, lateral septum labeling shows profiles of neuronal processes

• Subcellular Localization:

  • SYNGAP1 is primarily localized to the postsynaptic density in excitatory synapses

  • When interpreting immunofluorescence data, consider co-staining with synaptic markers to confirm proper localization

• Developmental Changes:

  • SYNGAP1 expression and localization change during development

  • When comparing across ages, consider that peak expression may occur during specific developmental windows associated with synaptogenesis

• Circadian Variation:

  • Consider potential circadian fluctuations in expression levels

  • Studies have examined SYNGAP1 expression at different circadian timepoints (CT6 and CT14)

  • When designing experiments, control for time of day to minimize variability

What are important considerations for quantitative analysis of SYNGAP1 levels?

Quantitative analysis of SYNGAP1 requires rigorous methodology:

• Western Blot Quantification:

  • Use consistent loading controls appropriate for brain tissue (e.g., β-actin, GAPDH)

  • For circadian studies or experiments comparing timepoints, consider pooling samples from multiple animals (e.g., 3 animals per sample)

  • Load consistent amounts of protein (e.g., 10 μg/lane)

• Immunohistochemical Quantification:

  • Use standardized image acquisition settings across all samples

  • For DAB-based detection, consider using nickel intensification for enhanced sensitivity

  • Analyze multiple sections per animal and multiple animals per condition

• Phosphorylation State Analysis:

  • When using phospho-specific antibodies like SynGAP pSer1138, normalize to total SYNGAP1 levels

  • Consider both absolute levels and phosphorylation ratios in your analysis

• Statistical Considerations:

  • Account for biological variability by using appropriate sample sizes

  • Consider the distribution of your data when selecting statistical tests

  • For developmental or circadian studies, control for time-dependent effects

How are SYNGAP1 antibodies contributing to our understanding of neurodevelopmental disorders?

SYNGAP1 antibodies are facilitating critical advances in understanding neurodevelopmental disorders:

• Autism Spectrum Disorder (ASD) Research:

  • Investigations have revealed that SYNGAP1 mutations can reshape its synaptic interactome

  • Studies using SYNGAP1 antibodies have identified that SYNGAP1 binding with AnkS1b is disrupted by an ASD-associated SYNGAP1 mutation

  • Markov cluster analysis of the SYNGAP1-Anks1b overlapping interactome identified a protein community significantly enriched for pathways of "autistic disorder" and "glutamate receptor signaling"

• Functional Consequences of Mutations:

  • Electrophysiological studies combined with antibody validation have shown that SYNGAP1 loss-of-function mutations atypically accelerate network activity specifically during synaptogenesis periods

  • Further depletion of Anks1b exacerbates the abnormal heightened neural activity linked to SYNGAP1 mutation

• Potential Therapeutic Targets:

  • Proteomic analysis using SYNGAP1 antibodies has helped identify targets that may restore spontaneous activity of neurons harboring patient-derived mutations

  • The physical and functional interaction between SYNGAP1 and Anks1b within a common pathway regulating neuronal activity development suggests potential therapeutic avenues

What novel techniques are being developed for studying SYNGAP1 protein complexes?

Innovative techniques for studying SYNGAP1 protein complexes include:

• High-throughput In Vivo Biotinylation Approaches:

  • HiUGE-iBioID techniques have been employed to identify proteins interacting with SYNGAP1

  • These approaches revealed that 94% of SYNGAP1-interacting proteins were also found in the Anks1b interactome

• Chemico-genetic Analysis:

  • Chemico-genetic analysis of native autism proteomes has revealed functional interactions and mechanisms underlying ASD-associated proteins

  • This approach has identified modules involved in actin regulation in SYNGAP1 networks

• Spatial Co-perturbation Proteomics:

  • This technique combines spatial information with perturbation-based proteomics

  • It has been applied to identify targets that can restore spontaneous activity of neurons harboring patient-derived mutations

• Biotinylated RNA Pull-down Assays:

  • These assays assess the interaction of SYNGAP1 mRNA 3'UTR with endogenous RNA-binding proteins

  • They have been used to investigate how UTR variants cause aberrant SYNGAP1 splicing in ALS patients

How can researchers integrate SYNGAP1 antibody data with other omics approaches?

Integrating SYNGAP1 antibody data with other omics approaches can provide comprehensive insights:

• Multi-omics Integration:

  • Combine protein-level data from SYNGAP1 antibody studies with transcriptomic data to correlate protein expression with mRNA levels

  • Integrate with phosphoproteomic data to understand post-translational modifications

  • Correlate with electrophysiological data to link molecular changes to functional outcomes

• Network Analysis:

  • Use Markov cluster (MCL) analysis of interactome data to identify protein communities

  • Research has identified a large PPI community (32 of 92 proteins) including SYNGAP1 and Anks1b that is significantly enriched for pathways relevant to neurological disorders

• Temporal Dynamics:

  • Combine antibody-based protein quantification with longitudinal electrophysiological monitoring

  • This approach has revealed developmental timepoint-specific effects of SYNGAP1 mutations on neural activity

• Functional Validation:

  • Use CRISPR-based approaches in combination with antibody validation to study the effects of genetic manipulations

  • AAV-mediated CRISPR disruption targeting SYNGAP1 at specific exons has been used to achieve protein loss and study functional consequences