STT3B Antibody, HRP conjugated

What is STT3B and what is its functional significance in cellular physiology?

STT3B (STT3 oligosaccharyltransferase complex catalytic subunit B) is a critical component of the N-oligosaccharyl transferase (OST) complex that catalyzes the initial transfer of a defined glycan (Glc₃Man₉GlcNAc₂ in eukaryotes) from dolichol-pyrophosphate to asparagine residues within Asn-X-Ser/Thr consensus motifs in nascent polypeptide chains . This represents the first and rate-limiting step in protein N-glycosylation.

The human STT3B protein consists of 826 amino acid residues with a molecular mass of approximately 93.7 kDa . It is primarily localized in the endoplasmic reticulum and is widely expressed across multiple tissues including heart, brain, placenta, lung, liver, muscle, kidney, and pancreas .

Unlike its paralogue STT3A, which primarily functions cotranslationally, STT3B-containing complexes have specialized functions:

• They can mediate glycosylation of nascent sites inaccessible to STT3A

• They operate post-translationally to modify skipped glycosylation sites in unfolded proteins

• They play a critical role in ER-associated degradation (ERAD) by glycosylating misfolded proteins, facilitating their recognition and subsequent degradation

What applications are STT3B antibodies typically used for in research settings?

STT3B antibodies serve multiple research applications across different experimental techniques:

The specific applications depend on the research question being addressed . When selecting an STT3B antibody, researchers should verify the validated applications listed by the manufacturer.

How do STT3A and STT3B functions differ in the glycosylation process?

STT3A and STT3B represent two distinct catalytic subunits of the OST complex with different functional properties:

STT3B seems to act as a "backup" system to glycosylate sites that STT3A fails to modify or cannot access . Additionally, STT3B plays a significant role in marking misfolded proteins through glycosylation for subsequent degradation, a crucial aspect of ER quality control .

What are the optimal protocols for using HRP-conjugated STT3B antibodies in ELISA?

When utilizing HRP-conjugated STT3B antibodies in ELISA applications, consider the following optimization protocol:

Recommended ELISA Protocol:

• Coating: Coat high-binding 96-well plates with capture antibody or antigen at 1-10 μg/mL in carbonate buffer (pH 9.6) overnight at 4°C

• Blocking: Block with 3-5% BSA or 5% non-fat milk in PBS or TBS for 1-2 hours at room temperature

• Sample incubation: Apply diluted samples and standards in blocking buffer for 2 hours at room temperature

• Detection: Dilute HRP-conjugated STT3B antibody (typically 1:1000 to 1:5000) in blocking buffer and incubate for 1-2 hours

• Substrate reaction: Add TMB substrate and incubate for 15-30 minutes in the dark

• Stop reaction: Add 2N H₂SO₄ and read absorbance at 450 nm

Optimization considerations:

• Perform antibody titration to determine optimal concentration (usually 0.1-1.0 μg/mL)

• Include proper controls: positive control (recombinant STT3B), negative control, and background control

• For detection of STT3B protein, sandwich ELISA using a capture antibody recognizing a different epitope is recommended

• Store HRP-conjugated antibodies at -80°C in single-use aliquots to maintain enzymatic activity

How can STT3B antibodies be utilized to investigate post-translational glycosylation in research models?

STT3B's distinct role in post-translational glycosylation makes antibodies against this protein valuable tools for investigating various aspects of this process:

Experimental Approach for Studying Post-Translational Glycosylation:

• Comparative analysis of STT3A vs. STT3B-mediated glycosylation:

  • Use siRNA knockdown or CRISPR-Cas9 knockout of STT3A or STT3B separately

  • Employ STT3B antibodies in Western blot to confirm knockdown/knockout efficiency

  • Analyze glycosylation patterns using glycan-specific lectins or antibodies

• Identification of STT3B-specific substrates:

  • Immunoprecipitate STT3B using specific antibodies

  • Perform mass spectrometry to identify interacting proteins

  • Validate interactions using co-immunoprecipitation followed by Western blot

• Monitoring STT3B involvement in ERAD pathway:

  • Create reporter constructs with known ERAD substrates

  • Track their degradation in the presence/absence of STT3B

  • Use HRP-conjugated STT3B antibodies in pulse-chase experiments to monitor protein turnover

• Visualization of STT3B distribution during stress conditions:

  • Induce ER stress using tunicamycin or thapsigargin

  • Perform immunofluorescence with STT3B antibodies to track redistribution

  • Co-stain with other ER stress markers to correlate STT3B localization with stress response

This methodological approach allows researchers to dissect the specific contributions of STT3B to post-translational glycosylation and protein quality control.

What methodological approaches can resolve contradictory findings in STT3B-related glycosylation research?

Contradictory findings in STT3B research may stem from various factors including cell type specificity, experimental conditions, and antibody characteristics. The following methodological approaches can help resolve such discrepancies:

• Antibody validation strategy:

  • Confirm antibody specificity using multiple methods: Western blot, immunoprecipitation, and immunofluorescence

  • Validate results using multiple antibodies targeting different epitopes of STT3B

  • Include proper controls: STT3B knockout/knockdown cells, blocking peptides, and isotype controls

• Comprehensive glycosylation analysis:

  • Combine multiple techniques: mass spectrometry, lectin binding assays, and glycosidase treatments

  • Compare site-specific glycosylation using glycopeptide enrichment followed by LC-MS/MS

  • Analyze glycan structures using exoglycosidase digestions and HILIC separation

• Cell-type specific considerations:

  • Systematically compare STT3B function across different cell types

  • Account for tissue-specific expression patterns of auxiliary OST components

  • Consider the influence of cell-specific glycosylation machinery

• Temporal analysis of glycosylation:

  • Utilize pulse-chase experiments to distinguish cotranslational vs. post-translational events

  • Implement time-resolved studies using synchronized protein expression systems

  • Employ real-time imaging with fluorescently-tagged STT3B to track dynamic changes

By implementing these approaches, researchers can develop a more nuanced understanding of the context-dependent functions of STT3B in glycosylation processes.

How can STT3B antibodies contribute to investigating viral glycoprotein processing?

Recent research has revealed that STT3B plays a critical role in the glycosylation of viral proteins, making STT3B antibodies valuable tools for studying virus-host interactions:

Experimental Framework for Viral Glycoprotein Research:

• Virus-specific STT3B interactions:

  • Research indicates that Lassa virus (LASV) glycoprotein (GP) is preferentially modified by the STT3B-OST isoform rather than STT3A

  • Use co-immunoprecipitation with STT3B antibodies to pull down viral glycoproteins

  • Perform reverse immunoprecipitation with viral protein antibodies to confirm interactions

• Functional impact analysis:

  • Compare viral replication in STT3B-knockdown versus control cells

  • Assess viral glycoprotein maturation using pulse-chase experiments

  • Examine changes in viral protein stability and trafficking

• Visualization of virus-induced STT3B redistribution:

  • Track STT3B localization during viral infection using immunofluorescence

  • Co-stain with viral proteins to identify colocalization patterns

  • Use super-resolution microscopy to examine nanoscale organization

• Therapeutic implications:

  • Screen for compounds that disrupt STT3B-viral glycoprotein interactions

  • Use STT3B antibodies to monitor binding inhibition

  • Develop assays to identify novel antiviral targets based on STT3B function

This approach leverages STT3B antibodies to gain insights into virus-host interactions that may lead to the development of novel therapeutic strategies against viruses like Lassa that depend on STT3B-mediated glycosylation.

What are the critical factors for optimizing Western blot detection using STT3B antibodies?

Successful Western blot detection of STT3B requires careful optimization due to its high molecular weight (~94 kDa) and membrane protein characteristics:

Optimized Western Blot Protocol for STT3B Detection:

• Sample preparation:

  • Use strong lysis buffers containing 1% SDS or RIPA buffer with protease inhibitors

  • Avoid boiling samples (heat at 70°C for 10 minutes instead)

  • Include reducing agents (DTT or β-mercaptoethanol) in sample buffer

• Gel electrophoresis considerations:

  • Use 8% acrylamide gels or gradient gels (4-15%) to better resolve high molecular weight proteins

  • Load adequate protein amount (30-50 μg of total protein)

  • Include positive control (recombinant STT3B or lysate with known STT3B expression)

• Transfer parameters:

  • Perform wet transfer at 30V overnight at 4°C for efficient transfer of high molecular weight proteins

  • Use PVDF membranes with 0.45 μm pore size rather than 0.2 μm

  • Add 0.1% SDS to transfer buffer to aid in transfer of hydrophobic proteins

• Antibody incubation:

  • Block with 5% BSA in TBST (not milk, which can interfere with phospho-detection)

  • Use primary antibody at 1:500-1:1000 dilution overnight at 4°C

  • For HRP-conjugated antibodies, optimize concentration to reduce background

  • Include longer washing steps (5 × 5 minutes) to reduce background

• Detection system:

  • Use enhanced chemiluminescence with extended exposure times if necessary

  • Consider signal enhancers for weak signals

  • For challenging samples, try fluorescent secondary antibodies and imaging systems

If using HRP-conjugated primary antibodies directly, ensure they maintain enzymatic activity and perform additional blocking steps to minimize non-specific binding.

How can researchers validate the specificity of STT3B antibodies for their experimental system?

Rigorous validation of STT3B antibodies is essential for generating reliable research data. Implement the following comprehensive validation strategy:

• Genetic validation approaches:

  • Test antibody in STT3B knockout/knockdown models (siRNA, shRNA, or CRISPR-Cas9)

  • Overexpress tagged STT3B constructs and confirm co-detection with tag-specific antibodies

  • Perform rescue experiments to confirm specificity of observed effects

• Biochemical validation methods:

  • Use epitope blocking peptides to confirm binding specificity

  • Compare multiple antibodies targeting different regions of STT3B

  • Perform immunoprecipitation followed by mass spectrometry identification

• Cross-reactivity assessment:

  • Test antibody reactivity in samples expressing only STT3A but not STT3B

  • Evaluate potential cross-reactivity with other OST complex components

  • Assess species specificity through comparative analysis across different organisms

• Application-specific validation:

  • For immunohistochemistry: compare with in situ hybridization or RNAscope

  • For immunofluorescence: co-stain with established ER markers

  • For flow cytometry: use isotype controls and secondary-only controls

Proper validation ensures that experimental results accurately reflect STT3B biology rather than artifacts of non-specific antibody interactions.

How are STT3B antibodies being used to investigate the role of glycosylation in disease pathogenesis?

STT3B antibodies are increasingly employed to explore the connections between aberrant glycosylation and disease mechanisms:

Disease-Focused Research Applications:

• Cancer biology:

  • Monitor STT3B expression across tumor types and stages

  • Correlate glycosylation patterns with disease progression

  • Investigate STT3B-mediated glycosylation of tumor-associated antigens

• Neurodegenerative disorders:

  • Study STT3B's role in processing disease-related proteins

  • Research indicates STT3B mediates glycosylation of the disease variant AMYL-TTR 'Asp-38' of TTR at 'Asn-118', facilitating its degradation

  • Investigate potential therapeutic approaches targeting STT3B function

• Viral pathogenesis:

  • Examine STT3B interactions with viral glycoproteins

  • Lassa virus glycoprotein (LASV GP) is preferentially modified by the STT3B-OST isoform, suggesting a critical role in viral infection

  • Develop novel antiviral strategies based on STT3B inhibition

• Congenital disorders of glycosylation:

  • Assess STT3B mutations or expression changes in patient samples

  • Study compensatory mechanisms between STT3A and STT3B

  • Develop diagnostic tools using STT3B antibodies

These research directions highlight the expanding role of STT3B antibodies in understanding disease mechanisms linked to protein glycosylation defects.

What are the latest methodological advances in using STT3B antibodies for integrative glycoproteomics?

Recent technological developments have enhanced the utility of STT3B antibodies in comprehensive glycoproteomic analyses:

• Proximity labeling approaches:

  • STT3B-BioID or APEX2 fusion proteins to identify proximal interacting partners

  • Spatial mapping of the glycosylation machinery in the ER

  • Temporal analysis of dynamic interactions during stress conditions

• Single-cell glycoproteomics:

  • Integration of STT3B antibodies with mass cytometry (CyTOF)

  • Correlation of STT3B expression with cell-specific glycosylation patterns

  • Development of multiplexed glycoprotein detection systems

• Glycosite-specific antibody development:

  • Generation of antibodies that recognize specific glycosylated versus non-glycosylated sites

  • Analysis of site occupancy in STT3B-dependent glycosylation

  • Quantitative assessment of glycosylation efficiency

• Combined proteome and glycoproteome analyses:

  • Integrated workflow using STT3B antibodies for protein enrichment

  • Parallel analysis of protein expression and glycosylation status

  • Bioinformatic integration of proteomic and glycomic datasets to identify STT3B-specific substrates

These methodological advances represent the cutting edge of glycobiology research and offer powerful new tools for investigating the complex roles of STT3B in cellular physiology and disease.