SYVN1 Antibody, Biotin conjugated

What is SYVN1 and what are its primary functions in cellular processes?

SYVN1 (Synovial apoptosis inhibitor 1), also known as HRD1, is an E3 ubiquitin-protein ligase that plays critical roles in cellular protein quality control. It specifically accepts ubiquitin from endoplasmic reticulum-associated UBC7 E2 ligase and transfers it to substrate proteins, promoting their degradation .

SYVN1 functions include:

• Serving as a key component of the endoplasmic reticulum quality control (ERQC) system, also called ER-associated degradation (ERAD)

• Mediating ubiquitin-dependent degradation of misfolded endoplasmic reticulum proteins

• Promoting degradation of normal but naturally short-lived proteins

• Protecting cells from ER stress-induced apoptosis

• Protecting neurons from apoptosis induced by polyglutamine-expanded huntingtin or unfolded GPR37

SYVN1 is ubiquitously expressed, with highest levels found in liver and kidney tissues, and is upregulated in synovial tissues from patients with rheumatoid arthritis .

How can I optimize immunoprecipitation protocols when using biotin-conjugated SYVN1 antibodies?

When optimizing immunoprecipitation (IP) with biotin-conjugated SYVN1 antibodies, follow these methodological approaches:

• Sample preparation:

  • For tissue samples like dorsal striatum, homogenize in appropriate lysis buffer containing protease inhibitors

  • For cell cultures, lyse cells directly in non-denaturing IP buffer

• Antibody binding:

  • Use appropriate dilution (typically 1:100-1:200) based on biotin-conjugated antibody concentration

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate lysates with biotin-conjugated SYVN1 antibody overnight at 4°C

• Precipitation method:

  • Use streptavidin-coated magnetic beads rather than agarose for higher purity

  • Employ gentle washing steps (at least 3-5 washes) with buffer containing 0.1-0.5% detergent

• Controls to include:

  • Input control (5-10% of starting material)

  • IgG control to assess non-specific binding

  • Known SYVN1 interaction partner as positive control

This approach has been successfully used to demonstrate SYVN1's interaction with substrates such as GABA Aα1, as shown by co-immunoprecipitation experiments where "SYVN1 was detected in GABA Aα1 immunoprecipitates, but not in the control IgG" .

What are the most effective methods for studying SYVN1-mediated protein degradation pathways?

To effectively study SYVN1-mediated protein degradation pathways, researchers should employ a multi-faceted approach:

• Genetic manipulation of SYVN1 expression:

  • RNA interference: Use SYVN1-specific shRNA delivered via lentiviral vectors (Lenti-SYVN1) for in vitro studies or adeno-associated viral vectors (AAV-SYVN1) for in vivo studies

  • Quantify knockdown efficiency via western blotting (typically achieving 60-70% reduction)

• Proteasome inhibition studies:

  • Use specific inhibitors such as MG132 and Lactacystin

  • Monitor accumulation of SYVN1 substrates following inhibitor treatment

  • Compare effects of proteasome inhibition versus SYVN1 knockdown

• Tracking substrate degradation:

  • Pulse-chase experiments with radiolabeled substrates

  • Cycloheximide chase assays to monitor substrate half-life

  • Compare degradation kinetics between wildtype and SYVN1-deficient conditions

• Ubiquitination analysis:

  • Immunoprecipitate substrate proteins and blot for ubiquitin

  • Use ubiquitin mutants (K48R, K63R) to determine ubiquitin chain topology

  • Research has shown that "under the catalysis of SYVN1, the conjugation efficiency of WT ubiquitin or ubiquitin containing only K48 to PPARα was high, while the conjugation efficiency of ubiquitin containing only K63 to PPARα was low"

• Compartment-specific analysis:

  • Fractionate cells to separate intra-ER from extra-ER components

  • Compare substrate levels between compartments following SYVN1 manipulation

  • Research demonstrates that "SYVN1 knockdown increased GABA Aα1 in the intra-ER, but not in the extra-ER"

How can researchers distinguish between SYVN1's role in proteasomal degradation versus selective autophagy?

Distinguishing between SYVN1's involvement in proteasomal degradation versus selective autophagy requires specific experimental approaches:

• Comparative inhibition studies:

  • Proteasome inhibitors: MG132, lactacystin, bortezomib

  • Autophagy inhibitors: Bafilomycin A1 (Baf A1), NH₄Cl, chloroquine

  • Monitor substrate accumulation under each condition

• Genetic manipulation:

  • Compare effects of SYVN1 knockdown in wildtype versus autophagy-deficient cells (e.g., atg5^-/-^ MEFs)

  • Research has shown that "SYVN1 dramatically decreased insoluble SERPINA1 E342K/ATZ levels in WT MEFs, but SYVN1 hardly affected insoluble SERPINA1 E342K/ATZ levels in atg5 knockout MEFs"

• Ubiquitin chain analysis:

  • K48-linked chains: typically target proteins for proteasomal degradation

  • K63-linked chains: often associated with autophagy

  • Use chain-specific antibodies or ubiquitin mutants (K48R, K63R)

  • Studies indicate SYVN1 "predominantly conjugates K48-linked polyubiquitin chains to PPARα" and "SYVN1-mediated lysine 48 (K48)-linked polyubiquitin chains that conjugated onto SERPINA1 E342K/ATZ might predominantly bind to the ubiquitin-associated (UBA) domain of SQSTM1"

• Co-localization studies:

  • Assess co-localization with autophagy markers (LC3, SQSTM1/p62)

  • Monitor formation of autophagosomes/autolysosomes

  • Research demonstrates that "SYVN1 increased the colocalization between SERPINA1 E342K/ATZ and LAMP1 (lysosomal-associated membrane protein 1), the major lysosomal membrane glycoprotein"

• Solubility fractionation:

  • Separate soluble from insoluble protein fractions

  • Research shows "SYVN1 is mainly responsible for disposal of insoluble SERPINA1 E342K/ATZ via the autophagy pathway"

What are the optimal dilution ratios and incubation conditions for biotin-conjugated SYVN1 antibodies in different applications?

Based on established protocols, the following parameters are recommended for biotin-conjugated SYVN1 antibodies:

For sandwich ELISA protocols specifically:

• Coat plate with capture antibody

• Add samples and standards

• Add biotin-conjugated detection antibody (1:100 dilution)

• Add Streptavidin-HRP (1:100 dilution)

• Add substrate solution and measure at 450nm

"Prepare biotinylated antibody working solution within 1 hour before experiment. Calculate required total volume: 0.1 ml/well × quantity of wells (plus 0.1-0.2 ml extra). Dilute the Biotinylated antibody with Biotin Antibody Dilution Buffer at 1:100" .

How can researchers effectively validate SYVN1-substrate interactions in different cellular compartments?

To effectively validate SYVN1-substrate interactions across different cellular compartments, employ these methodological approaches:

• Subcellular fractionation:

  • Separate cellular components (ER, cytosol, membrane, nucleus)

  • Use ultracentrifugation with sucrose gradients for higher resolution

  • Validate fraction purity with compartment-specific markers:

    • ER: Calnexin, PDI

    • Cytosol: GAPDH, tubulin

    • Membrane: Na+/K+ ATPase

    • Nucleus: Lamin B1

• Compartment-specific co-immunoprecipitation:

  • Perform co-IP from isolated subcellular fractions

  • Compare interaction patterns between compartments

  • Include appropriate controls for each fraction

  • Research demonstrated this approach by showing "SYVN1 knockdown increased GABA Aα1 in the intra-ER, but not in the extra-ER"

• Proximity ligation assay (PLA):

  • Visualize and quantify protein-protein interactions in situ

  • Combine with compartment-specific markers for spatial resolution

  • Provides single-molecule sensitivity

• Fluorescence microscopy techniques:

  • Co-localization analysis with compartment markers

  • FRET (Förster Resonance Energy Transfer) for direct interaction

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility

• Domain mapping of interaction sites:

  • Generate deletion constructs to identify binding domains

  • Research has identified specific binding sites, for example "three binding sites between GLI2 and SYVN1 were predicted"

How should researchers approach studying SYVN1's role in neurodegenerative disease models?

When investigating SYVN1's role in neurodegenerative disease models, researchers should implement the following methodological approach:

• Selection of appropriate models:

  • Cellular models: Primary neurons, neuronal cell lines, patient-derived iPSCs

  • Animal models: Transgenic mice, conditional knockouts, viral-mediated gene transfer

  • Research has successfully used "rat METH conditioned place preference (CPP) model" to study SYVN1's role in GABA Aα1 degradation

• Viral vector-based manipulation strategies:

  • For in vitro studies: Lentiviral vectors (e.g., Lenti-SYVN1)

  • For in vivo studies: Adeno-associated viral vectors (e.g., AAV-SYVN1)

  • Research has demonstrated "infection of primary Dstr neurons with Lenti-SYVN1 significantly decreased SYVN1 expression level" and "infection of striatum neurons with AAV-SYVN1 significantly decreased SYVN1 expression level"

• Behavioral assessment combined with molecular analysis:

  • Correlate behavioral changes with molecular alterations

  • Assess SYVN1 activity in affected brain regions

  • Research has shown that "METH-induced CPP formation was accompanied by a significant increase in the expression of SYVN1 in the Dstr"

• Substrate identification and validation:

  • Proteomics approach to identify disease-specific substrates

  • Validate using co-IP, proximity ligation assays

  • Research demonstrated "GABA Aα1 interacted with SYVN1 in the Dstr of METH pairing rat"

• Therapeutic potential assessment:

  • Evaluate whether SYVN1 modulation affects disease progression

  • Consider both inhibition and enhancement strategies

  • Research suggests SYVN1 as "a promising therapeutic target" for various diseases

What experimental approaches can best elucidate the mechanistic role of SYVN1 in protein quality control during cellular stress response?

To effectively investigate SYVN1's mechanistic role in protein quality control during stress response, researchers should employ these approaches:

• Controlled induction of cellular stress:

  • ER stress: Tunicamycin, thapsigargin, DTT

  • Oxidative stress: H₂O₂, paraquat

  • Inflammatory stress: LPS, TNF-α

  • Disease-specific stressors: "LPS-exposed cells" have been used to study SYVN1 in septic liver injury

• Time-course analysis of SYVN1 activation:

  • Monitor SYVN1 expression, localization, and activity at multiple timepoints

  • Track ER stress markers simultaneously (GRP78/BiP, CHOP)

  • Research has shown that "endoplasmic reticulum stress (ERS)-associated Glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) increased" following SYVN1 knockdown

• Substrate fate mapping:

  • Pulse-chase studies to track substrate degradation kinetics

  • Ubiquitination profiling under different stress conditions

  • Polyubiquitin chain topology analysis (K48 vs. K63 linkages)

  • Research demonstrated "SYVN1 predominantly conjugates K48-linked polyubiquitin chains to PPARα"

• Mechanistic dissection using domain mutants:

  • RING domain mutants (e.g., SYVN1C1A) to disrupt E3 activity

  • Transmembrane domain mutations to alter ER localization

  • Studies utilized "E3 activity-deficient mutant SYVN1C1A plasmid (a mutation in the first 2 zinc-coordinating cysteine residues in the RING finger domain)"

• Integration with other quality control pathways:

  • Examine cross-talk between ERAD, UPR, and autophagy

  • Research has demonstrated that "Normally, GABA Aα1 proteins was correctly folded in ER, and then exported to cytosol or assembled to the cell membrane. METH treatment caused misfolded or inappropriate GABA Aα1 accumulated in the ER, induced ERS and led to increase of GRP78 to help to modifying misfolded GABA Aα1 proteins. Misfolded GABA Aα1 proteins are then delivered to SYVN1 and degraded by UPS"

How can researchers effectively quantify changes in SYVN1-mediated ubiquitination across experimental conditions?

To accurately quantify changes in SYVN1-mediated ubiquitination across experimental conditions, researchers should employ these quantitative approaches:

• In vitro ubiquitination assays:

  • Reconstitute ubiquitination reaction with purified components:

    • E1 (ubiquitin-activating enzyme)

    • E2 (UBC7/UBE2G2)

    • SYVN1 (E3)

    • Substrate protein

    • Ubiquitin (wild-type or mutant)

  • Quantify ubiquitin incorporation by western blotting

  • Compare wild-type SYVN1 with catalytically inactive mutants (C1A)

• Ubiquitin chain topology analysis:

  • Use ubiquitin mutants (K48R, K63R) to determine linkage specificity

  • Apply ubiquitin chain-specific antibodies for detection

  • Research shows "under the catalysis of SYVN1, the conjugation efficiency of WT ubiquitin or ubiquitin containing only K48 to PPARα was high, while the conjugation efficiency of ubiquitin containing only K63 to PPARα was low"

• Tandem ubiquitin binding entities (TUBEs):

  • Enrich for ubiquitinated proteins under native conditions

  • Preserve ubiquitin chain integrity during isolation

  • Quantify by western blotting or mass spectrometry

• Mass spectrometry-based approaches:

  • Absolute quantification using isotope-labeled standards

  • SILAC or TMT labeling for relative quantification

  • Identification of specific ubiquitination sites (K-GG peptides)

• Cellular ubiquitination reporters:

  • Fluorescent ubiquitination-based cell cycle indicator (FUCCI)

  • Bioluminescence resonance energy transfer (BRET)-based sensors

  • Develop substrate-specific reporters for real-time monitoring

Researchers should normalize data to appropriate controls and perform statistical analysis to determine significant changes in ubiquitination levels across experimental conditions.

What are the most effective strategies for developing SYVN1-targeted therapeutic approaches for neurodegenerative and inflammatory diseases?

For developing effective SYVN1-targeted therapeutic approaches, researchers should consider these methodological strategies:

• Target validation in disease-relevant models:

  • Genetic manipulation in animal models of disease

  • Patient-derived cellular systems (iPSCs, organoids)

  • Correlation studies in human pathological samples

  • Research has identified SYVN1 as "a promising therapeutic target for various diseases"

• Small molecule inhibitor development pipeline:

  • High-throughput screening of compound libraries

  • Structure-based drug design targeting SYVN1's RING domain

  • Allosteric modulators affecting substrate binding

  • Phenotypic screening in disease models

• Substrate-specific intervention strategies:

  • Identify disease-specific SYVN1 substrates

  • Develop molecules that selectively disrupt SYVN1-substrate interactions

  • Design peptide-based inhibitors mimicking substrate binding regions

  • Research has identified various substrates including "GABA Aα1" , "PPARα" , and "SERPINA1 E342K/ATZ"

• Gene therapy approaches:

  • AAV-mediated delivery of SYVN1 modulators

  • CRISPR-based editing of SYVN1 or substrate proteins

  • Regulatable expression systems for timing control

  • Research has successfully used "AAV-SYVN1" for in vivo manipulation

• Combination strategies targeting interconnected pathways:

  • Co-targeting UPR components alongside SYVN1

  • Modulators of autophagy to complement SYVN1 targeting

  • Anti-inflammatory approaches for diseases with inflammatory components

  • Research shows "autophagy induction enhanced SYVN1-mediated SERPINA1 E342K/ATZ degradation"