SYVN1 Antibody, HRP conjugated

What is SYVN1 and why is it an important research target?

SYVN1 (Synovial Apoptosis Inhibitor 1, also known as Synoviolin or HRD1) is a RING-type E3 ubiquitin ligase primarily localized to the endoplasmic reticulum. It plays crucial roles in multiple cellular processes including:

• Endoplasmic reticulum-associated degradation (ERAD)

• Regulation of pyroptosis through GSDMD ubiquitination

• Control of airway remodeling in asthma via SIRT2 degradation

• GABA receptor degradation in neurological processes

• Regulation of protein homeostasis through both proteolytic and non-proteolytic ubiquitination

The study of SYVN1 provides insights into cellular stress responses, inflammatory mechanisms, and protein quality control pathways, making it relevant to research across immunology, neuroscience, and respiratory medicine .

What are the recommended protocols for using HRP-conjugated SYVN1 antibody in Western blotting?

For optimal Western blot results with HRP-conjugated SYVN1 antibody:

Sample Preparation:

• Prepare total protein lysates from tissue or cells of interest

• Load 10-20 μg of total protein per lane

Dilution Recommendations:

• For rabbit polyclonal HRP-conjugated SYVN1 antibody: 1:100-1:500 for Western blotting

• For unconjugated primary antibodies followed by secondary detection: 1:1000-1:4000

Detection:

• Develop using ECL (Enhanced Chemiluminescence)

• Expected molecular weight: 67-76 kDa

Validation Controls:

• Positive controls: HEK-293, HeLa, HepG2, and Ramos cell lysates

• SYVN1 knockout/knockdown samples for specificity verification

The antibody shows reactivity with human, mouse, and rat samples, allowing for cross-species research applications .

How can I optimize immunofluorescence staining using SYVN1 antibodies?

For successful immunofluorescence with SYVN1 antibodies:

Cell Preparation:

• Grow cells on coverslips to 70-80% confluence

• Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes

Antibody Incubation:

• Block with 5% normal serum for 1 hour

• For HRP-conjugated antibodies: Convert to fluorescence using tyramide signal amplification

• For unconjugated primary antibodies: Use at 1:50-1:500 dilution

• Counterstain nuclei with DAPI

Visualization and Analysis:

• SYVN1 typically shows cytoplasmic localization with enrichment in the ER

• Co-staining with ER markers can confirm subcellular localization

Important Controls:

• Secondary antibody-only control to assess background

• SYVN1 knockdown cells to verify specificity

Notable colocalization studies have shown that SYVN1 interacts with GSDMD in the cytoplasm and with SIRT2 in cells, providing insights into its functional mechanisms .

How do I interpret contradictory results when studying SYVN1-mediated ubiquitination in different cellular contexts?

The interpretation of seemingly contradictory SYVN1-mediated ubiquitination results requires careful consideration of several factors:

Context-Dependent Effects:

• SYVN1 can mediate both proteolytic and non-proteolytic ubiquitination depending on substrate and cellular context

• In pyroptosis studies, SYVN1 promotes K27-linked polyubiquitination of GSDMD, enhancing rather than degrading its activity

• In contrast, SYVN1 facilitates degradation of SERPINA1E342K/ATZ through SQSTM1-dependent autophagy

Experimental Verification Approaches:

• Determine ubiquitination linkage types using linkage-specific antibodies

• Compare effects with SYVN1-C329S mutant (lacking E3 ligase activity)

• Use ubiquitin mutants with specific lysine residues (e.g., K27) to confirm linkage types

• Combine with proteasome inhibitors (MG132) or lysosome inhibitors (NH4Cl, Bafilomycin A1) to distinguish degradation pathways

Data Integration Strategy:
Create a comprehensive experimental design incorporating both gain-of-function (overexpression) and loss-of-function (SYVN1 knockout) approaches in parallel to resolve conflicting observations .

What are the critical considerations when designing co-immunoprecipitation experiments to study SYVN1 interactions with potential substrates?

To successfully identify and validate SYVN1-substrate interactions:

Experimental Design:

• Compare endogenous versus overexpression systems

  • Endogenous IP has been successful in THP-1 cells

  • Tagged overexpression systems work well in HEK293T cells

• Include bidirectional IP validation (IP with anti-SYVN1 and with anti-substrate antibodies)

Technical Optimizations:

• Use mild lysis conditions (1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions

• Add proteasome inhibitors (MG132, 10 μM for 4-6 hours) to stabilize transient interactions

• Include deubiquitinase inhibitors (N-ethylmaleimide, 10 mM) to preserve ubiquitination status

Controls and Validation:

• IgG control immunoprecipitation to assess non-specific binding

• SYVN1 mutant (SYVN1-C329S) lacking E3 ligase activity as a functional control

• Validation with alternative methods:

  • IP-LC-MS/MS for unbiased interaction discovery

  • Indirect immunofluorescence for colocalization confirmation

Published studies have successfully used these approaches to identify SYVN1 interactions with GSDMD, GABA A α1, and SIRT2, revealing its diverse regulatory functions .

How can I effectively analyze SYVN1-mediated K27-linked polyubiquitination versus other ubiquitin linkage types?

Distinguishing K27-linked polyubiquitination from other linkage types requires specialized approaches:

Experimental Strategy:

• Utilize ubiquitin mutants where only K27 is available for chain formation (other lysines mutated to arginine)

• Compare with other single-lysine ubiquitin mutants (K48, K63) to determine specificity

• Employ linkage-specific antibodies for direct detection of K27 chains

Technical Protocol:

• Co-transfect cells with:

  • Flag-tagged substrate (e.g., GSDMD)

  • HA-tagged ubiquitin or ubiquitin mutants

  • Myc-tagged SYVN1 or SYVN1-C329S

• Treat with proteasome inhibitor (10 μM MG132, 6 hours)

• Perform immunoprecipitation using anti-Flag antibody

• Probe western blots with:

  • Anti-HA to detect total ubiquitination

  • K27-linkage-specific antibody to detect specific chains

  • Anti-Flag to confirm substrate precipitation

Functional Validation:
Research has shown that K27-linked polyubiquitination by SYVN1 enhances GSDMD-mediated pyroptosis, differing from the conventional K48-linked degradative ubiquitination . This illustrates the importance of linkage-specific analysis in understanding SYVN1's diverse functions.

How can SYVN1 antibodies be applied to investigate endoplasmic reticulum stress in neurodegenerative disease models?

SYVN1 plays a critical role in ER stress regulation and neurodegeneration through several mechanisms:

Experimental Applications:

• Monitor SYVN1 expression changes during ER stress using HRP-conjugated antibodies in Western blot (1:1000 dilution)

• Track SYVN1-substrate interactions in neurodegenerative conditions

  • SYVN1 interacts with GABA A α1 in the striatum, affecting neuronal signaling

  • Changes in these interactions may indicate altered protein quality control

Protocol for ER Stress Analysis:

• Induce ER stress in neuronal cells using:

  • Thapsigargin (0.5-1 μM, 6-24 hours)

  • Tunicamycin (1-5 μg/ml, 6-24 hours)

• Analyze SYVN1 expression alongside ER stress markers:

  • GRP78/BiP, GRP94

  • p-PERK, p-IRE1, ATF6

• Perform co-immunoprecipitation to identify stress-dependent changes in SYVN1-substrate interactions

In Vivo Applications:
Research has demonstrated that SYVN1 interacts with GABA A α1 receptors in the striatum of rats, with altered interactions following methamphetamine conditioned pairing . This indicates potential roles in addiction and neuroplasticity mechanisms.

What methods should be used to evaluate SYVN1's role in pyroptosis and inflammatory disease models?

To investigate SYVN1's function in pyroptosis and inflammation:

Cell Culture Models:

• THP-1 monocytes (human) or primary macrophages

• HEK293T cells for reconstitution experiments

• Airway epithelial cells (BEAS-2B) for asthma models

Pyroptosis Induction Protocols:

• Canonical inflammasome activation:

  • LPS priming (1 μg/ml, 4 hours) followed by nigericin (5-10 μM, 1 hour)

• Non-canonical inflammasome activation:

  • Pam3CSK4 priming (1 μg/ml, 4 hours) followed by LPS transfection (1 μg/ml with lipofectamine)

Readout Measurements:

• LDH release assay to quantify cell death

• PI staining (red) for membrane permeabilization

• Western blot for GSDMD cleavage (detect p30 fragment)

• ELISA for IL-1β and IL-18 secretion

Genetic Manipulation Approaches:

• CRISPR/Cas9-mediated SYVN1 knockout

• Overexpression of wildtype SYVN1 vs. SYVN1-C329S (E3 ligase deficient mutant)

Research has demonstrated that SYVN1 significantly enhances pyroptosis through K27-linked polyubiquitination of GSDMD, making it a potential therapeutic target for inflammatory diseases .

How can I investigate SYVN1's role in asthma models using HRP-conjugated antibodies?

For studying SYVN1 in asthma pathophysiology:

Animal Model Protocol:

• OVA-induced murine asthma model:

  • Sensitization: OVA (20 μg) + aluminum hydroxide on days 0 and 14

  • Challenge: 1% OVA aerosol for 30 minutes on days 21, 22, and 23

Key Measurements:

• Lung histopathology (H&E, PAS, Masson staining)

• Airway inflammation and remodeling markers

• SYVN1 expression level changes

• ER stress markers (GRP78, GRP94, CHOP)

• EMT markers (E-cadherin, Vimentin)

SYVN1 Manipulation Approaches:

• Adenoviral-mediated SYVN1 overexpression in lungs

• Co-administration with SIRT2 to validate mechanistic pathway

Immunodetection Methods:

• Western blotting: Use HRP-conjugated SYVN1 antibody (1:1000 dilution)

• Immunohistochemistry: SYVN1 localization in bronchial epithelium

• Co-immunoprecipitation: SYVN1-SIRT2 interaction analysis

Research has shown that SYVN1 suppresses ER stress through ubiquitination and degradation of SIRT2, thereby protecting against airway remodeling in asthma models .

How can I address non-specific background when using HRP-conjugated SYVN1 antibodies in immunoblotting?

Excessive background when using HRP-conjugated SYVN1 antibodies can be resolved through several approaches:

Optimization Strategies:

• Titrate antibody concentration:

  • Start with higher dilutions (1:1000) and adjust as needed

  • Test multiple dilutions in parallel (1:500, 1:1000, 1:2000)

• Blocking optimization:

  • Try alternative blocking agents (5% BSA vs. 5% non-fat milk)

  • Extend blocking time to 2 hours at room temperature

• Washing optimization:

  • Increase washing duration (5 x 5 minutes with TBST)

  • Use 0.1% Tween-20 instead of 0.05% in wash buffer

Antibody-Specific Considerations:

• For HRP-conjugated antibodies: Add 0.05% sodium azide to the blocking buffer (NOT to the antibody solution as it inhibits HRP)

• Consider using Pierce™ Western Blot Signal Enhancer before antibody incubation

Sample Preparation Improvements:

• Include phosphatase inhibitors in lysis buffer to prevent non-specific bands

• Pre-clear lysates with Protein A/G beads before Western blotting

• Filter lysates through 0.45 μm filter to remove particulates

Comparing results across different SYVN1 antibodies (like those described in search results , , and ) can help confirm specific bands versus background.

What strategies can resolve inconsistent SYVN1 detection in different cell types and tissues?

When facing variable SYVN1 detection across samples:

Technical Adaptations:

• Sample-specific lysis optimization:

  • For tissues: Use RIPA buffer with mechanical disruption

  • For cells: NP-40 buffer may preserve protein interactions better

• Protein extraction enhancement:

  • Include 0.1% SDS in lysis buffer for membrane-associated proteins

  • For tissues: Homogenize with a Dounce homogenizer followed by sonication

Antibody Selection Strategy:

• For human samples: Anti-SYVN1 rabbit polyclonal shows consistent results

• For mouse/rat tissues: Verify species cross-reactivity in specific tissues

• Consider epitope location: C-terminal targeting antibodies detect most SYVN1 isoforms

Expression Level Considerations:

• SYVN1 expression varies by cell type and condition

• Highly expressed in: HEK293T, HeLa, HepG2 cells

• For low-expressing samples:

  • Increase protein loading (up to 50 μg)

  • Extend exposure time during chemiluminescence detection

  • Consider using SuperSignal West Femto substrate for enhanced sensitivity

Research shows that SYVN1 expression can be upregulated in certain conditions like OVA-induced asthma models or downregulated in others like TGF-β1-treated bronchial epithelial cells , explaining some detection variability.

What are the critical variables for reproducible co-immunoprecipitation experiments with SYVN1?

For consistent and reliable co-immunoprecipitation of SYVN1 with substrate proteins:

Critical Parameters:

• Cell lysis conditions:

  • Use gentle lysis buffers (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl pH 7.5)

  • Include freshly prepared protease inhibitor cocktail

  • For ubiquitination studies: Add deubiquitinase inhibitors (10 mM N-ethylmaleimide)

Antibody Selection:

• Verify antibody efficacy for IP applications

• For tagged proteins: Anti-tag antibodies often yield cleaner results than anti-SYVN1

• For endogenous IP: Use antibodies validated for immunoprecipitation applications (search result indicates successful IP with certain antibodies)

Technical Protocol Optimization:

• Pre-clear lysates with Protein A/G beads (1 hour at 4°C)

• Antibody binding: 3-4 μg antibody per 1 mg protein lysate, overnight at 4°C

• Bead washing: 5 washes with decreasing salt concentration

• Elution: Gentle elution with non-reducing sample buffer at 70°C (not 95°C)

Documentation for Reproducibility:

• Record exact protein concentrations in lysates

• Document incubation times precisely

• Maintain consistent temperature conditions throughout

• Include detailed controls in each experiment

Successful SYVN1 co-immunoprecipitation has been demonstrated with multiple substrates including GSDMD , GABA A α1 , and SIRT2 , providing validated protocol parameters.

How can SYVN1 antibodies be applied in studying non-proteolytic ubiquitination mechanisms?

SYVN1 has been implicated in both proteolytic and non-proteolytic ubiquitination, offering unique research opportunities:

Research Strategy:

• Distinguish non-proteolytic from degradative ubiquitination:

  • Monitor substrate levels over time after SYVN1 overexpression

  • Compare effects with and without proteasome/lysosome inhibitors

  • Analyze ubiquitin chain types (K27-linked for non-proteolytic functions)

Experimental Approaches:

• Ubiquitin Chain Analysis:

  • Utilize ubiquitin mutants (K27-only, K48-only, K63-only)

  • Use linkage-specific antibodies for chain type identification

  • Employ mass spectrometry to identify ubiquitination sites

• Functional Studies:

  • Compare wildtype SYVN1 with SYVN1-C329S (E3 ligase-dead mutant)

  • Assess different cellular outcomes beyond degradation:

    • Protein localization changes (e.g., MCT4 localization)

    • Activation/deactivation of enzymatic functions

    • Protein-protein interaction alterations

Recent research has revealed that SYVN1 mediates K27-linked polyubiquitination of GSDMD, enhancing rather than inhibiting its function in pyroptosis , and directs MCT4 localization through non-proteolytic ubiquitination .

What techniques can be combined with SYVN1 immunodetection to study its role in protein quality control pathways?

To comprehensively investigate SYVN1's function in protein quality control:

Integrated Approaches:

• Proximity Labeling Methods:

  • BioID or TurboID fused to SYVN1 to identify proximal interactors

  • APEX2-SYVN1 for temporal mapping of ERAD complexes

• Live Cell Imaging:

  • Fluorescently tagged SYVN1 combined with ER stress sensors

  • Photoactivatable SYVN1 to track substrate degradation kinetics

• Structural Analysis Techniques:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map SYVN1-substrate interfaces

  • Cryo-EM of SYVN1 complexes during ERAD

Example Combined Protocol for ERAD Substrate Tracking:

• Express fluorescently tagged ERAD substrate and SYVN1

• Induce misfolding with ER stressors (thapsigargin, tunicamycin)

• Track substrate localization and degradation via live imaging

• Fix cells at time points and perform immunostaining with HRP-conjugated SYVN1 antibody (1:100 dilution)

• Correlate imaging with biochemical assays (ubiquitination, degradation rate)

Studies have successfully applied these combined approaches to elucidate SYVN1's role in degrading misfolded proteins like SERPINA1E342K/ATZ via SQSTM1-dependent autophagy .

How can mass spectrometry be integrated with SYVN1 antibody techniques to identify novel substrates and interaction partners?

For comprehensive identification of SYVN1 substrates and interactors:

Integrated MS-Based Approaches:

• IP-MS Workflow:

  • Immunoprecipitate SYVN1 using validated antibodies

  • Analyze by LC-MS/MS to identify co-precipitated proteins

  • Validate hits with reciprocal co-IP and functional assays

• Ubiquitinome Analysis:

  • Compare ubiquitinated proteomes in SYVN1 wildtype vs. knockout cells

  • Enrich ubiquitinated peptides using K-ε-GG antibodies

  • Identify SYVN1-dependent ubiquitination sites

• Proximity-Based MS:

  • BioID-SYVN1 fusion to biotinylate proximal proteins

  • Streptavidin purification followed by MS analysis

  • Comparison across different cellular conditions

Experimental Design Considerations:

• Include MG132 treatment (10 μM, 4-6 hours) to stabilize transient interactions

• Compare results across multiple cell types (HEK293T, THP-1, tissue-specific cells)

• Use both C-terminal and N-terminal SYVN1 tagging to minimize functional interference

Validation Protocol:

• Select top candidates from MS analysis

• Perform targeted co-IP with SYVN1 antibodies

• Assess ubiquitination status with and without SYVN1 overexpression

• Determine functional outcomes (degradation, localization changes, activity modulation)

This integrated approach has successfully identified SYVN1 interaction with GSDMD , SIRT2 , and GABA A α1 , revealing its diverse regulatory functions across cellular processes.