SEC61B Antibody

What is SEC61B and what is its functional role in cellular processes?

SEC61B is the beta subunit of the Sec61 translocon complex located in the endoplasmic reticulum (ER) membrane. This protein plays a critical role in mediating membrane insertion of proteins associated with endocytosis and exocytosis pathways . The SEC61 complex forms a transmembrane channel through which newly synthesized proteins are translocated into the ER lumen or integrated into the ER membrane . SEC61B facilitates cotranslational protein transport across the ER membrane and has been shown to interact with signal recognition particles . At the molecular level, the gene encoding SEC61B has been associated with polycystic liver diseases, which are inherited disorders characterized by multiple hepatic cysts .

What types of SEC61B antibodies are available for research applications?

Current research-grade SEC61B antibodies fall into two main categories:

• Monoclonal antibodies: These include mouse anti-SEC61B antibodies like clone rAB01-4H3 (PrecisionAb Monoclonal), which offers high specificity and consistency between lots .

• Polyclonal antibodies: Examples include rabbit polyclonal antibodies (15087-1-AP and 51020-2-AP) that recognize different epitopes on the SEC61B protein .

The choice between monoclonal and polyclonal antibodies depends on experimental needs. Monoclonal antibodies provide higher specificity but may be more sensitive to epitope masking, while polyclonal antibodies offer broader epitope recognition but potentially higher background.

What applications can SEC61B antibodies be used for in research settings?

SEC61B antibodies have been validated for multiple research applications as shown in the following table:

Different antibodies show varying degrees of cross-reactivity with human, mouse, and rat samples, making them suitable for comparative studies across species .

How should I determine the optimal antibody concentration for detecting SEC61B in different cell lines?

Determining optimal antibody concentration requires systematic titration experiments. Begin with the manufacturer's recommended dilution range (e.g., 1:2000-1:12000 for Western blotting with 15087-1-AP ). Prepare a dilution series spanning this range and test against a constant amount of protein lysate from your target cell line.

For Western blot optimization:

• Load equal amounts of protein (20-30 μg) from your cell line of interest.

• Prepare 4-5 different antibody dilutions within the recommended range.

• Process identical blots with different antibody concentrations.

• Evaluate signal-to-noise ratio and specificity for each dilution.

• Select the dilution that provides clear detection of the expected 10-15 kDa band with minimal background.

Note that optimal concentration may vary between applications and cell lines. HepG2 cells consistently show strong SEC61B expression and can serve as a positive control .

What are the recommended sample preparation methods for detecting SEC61B in different experimental systems?

Sample preparation methods should be tailored to preserve SEC61B's native structure while maximizing extraction efficiency:

For Western blotting:

• Use RIPA buffer supplemented with protease inhibitors for whole-cell lysates.

• For membrane-enriched fractions, consider using subcellular fractionation to isolate ER membranes.

• Avoid extensive heating (>70°C) during sample denaturation as this may cause membrane protein aggregation.

• Use fresh samples when possible, as freeze-thaw cycles may affect membrane protein integrity.

For immunohistochemistry and immunofluorescence:

• For tissue sections, both paraformaldehyde fixation and paraffin embedding are suitable.

• Antigen retrieval is critical—use TE buffer at pH 9.0 (recommended) or citrate buffer at pH 6.0 as alternatives .

• For cultured cells, 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization generally yields good results.

Why might I observe multiple bands when probing for SEC61B in Western blots, and how can I verify antibody specificity?

Multiple bands in SEC61B Western blots may result from several factors:

• Post-translational modifications: SEC61B undergoes modifications that may alter its migration pattern.

• Protein degradation: Improper sample handling or insufficient protease inhibition.

• Non-specific binding: Particularly with polyclonal antibodies.

• Cross-reactivity with other SEC61 complex components: The alpha and gamma subunits have different molecular weights.

To verify antibody specificity:

• Compare observed molecular weight with expected size (10-15 kDa for SEC61B) .

• Use positive control lysates known to express SEC61B (e.g., HepG2, HeLa, HEK-293 cells) .

• Perform a blocking peptide competition assay using the immunogen peptide.

• Consider siRNA knockdown of SEC61B to confirm band identity.

• For definitive validation, use knockout cell lines as negative controls.

What are the common pitfalls when using SEC61B antibodies for immunofluorescence, and how can they be addressed?

Common pitfalls in SEC61B immunofluorescence include:

• High background staining:

  • Solution: Optimize blocking (use 5% BSA or 10% normal serum from the secondary antibody host species).

  • Increase washing steps (5 washes of 5 minutes each).

  • Further dilute primary antibody.

• Weak or no signal:

  • Solution: Ensure proper permeabilization (0.1-0.3% Triton X-100).

  • Optimize antigen retrieval for tissue sections.

  • Increase primary antibody concentration or incubation time (overnight at 4°C).

• Non-specific staining:

  • Solution: Pre-adsorb antibody with cell/tissue lysate from a non-relevant species.

  • Include additional blocking steps with normal serum.

• Autofluorescence interference:

  • Solution: Include Sudan Black B (0.1%) treatment to reduce autofluorescence.

  • Use appropriate filters to minimize autofluorescence detection.

• Incorrect subcellular localization:

  • Solution: Co-stain with established ER markers (e.g., calnexin, KDEL) to confirm proper localization.

  • Compare with published SEC61B localization patterns.

How can SEC61B antibodies be used to investigate changes in ER dynamics during cellular stress responses?

SEC61B antibodies can provide valuable insights into ER dynamics during stress conditions through several advanced applications:

• Quantitative Western blotting: To measure changes in SEC61B expression levels during ER stress. This approach can reveal adaptation mechanisms where translocon components are up- or down-regulated in response to unfolded protein response (UPR) activation.

• Co-immunoprecipitation (Co-IP): Using SEC61B antibodies for Co-IP (e.g., with 15087-1-AP ) allows identification of stress-specific interacting partners that may regulate translocon activity or composition during ER stress.

• Immunofluorescence with super-resolution microscopy: To visualize changes in SEC61B distribution and clustering during stress responses. This technique can reveal:

  • Translocon reorganization during ER expansion

  • Segregation of translocon components into specialized ER subdomains

  • Colocalization with stress-response proteins (e.g., BiP, PERK, IRE1)

• Live-cell imaging: In combination with SEC61B-GFP fusion proteins, antibody-based validation can confirm the physiological relevance of observed dynamics.

• Proximity ligation assay (PLA): To detect stress-induced changes in SEC61B's interaction with other translocon components or ER stress sensors with single-molecule sensitivity.

What approaches can be used to study SEC61B's role in protein translocation using available antibodies?

Several sophisticated approaches can be employed to investigate SEC61B's functional role in protein translocation:

• In vitro translation/translocation assays with SEC61B depletion: Deplete SEC61B from membrane fractions and assess the impact on translocation efficiency of model substrates. Verification of SEC61B depletion requires sensitive and specific antibodies.

• Immunodepletion and reconstitution experiments: Use SEC61B antibodies to immunodeplete the protein from cellular extracts, then reconstitute activity with recombinant SEC61B to confirm specificity.

• Site-specific crosslinking followed by immunoprecipitation: To map the interactions between SEC61B and translocation substrates at different stages of the translocation process.

• Antibody-based inhibition of translation/translocation: Application of Fab fragments of SEC61B antibodies to in vitro systems to determine if they directly interfere with translocation activity.

• Ribosome profiling with SEC61B immunoprecipitation: To identify transcripts actively engaged with SEC61B-containing translocons.

• CRISPR/Cas9 gene editing with antibody validation: Generate SEC61B variants and use antibodies to confirm expression and assess effects on translocon assembly and function.

How can SEC61B antibodies be used to investigate the pathogenesis of polycystic liver diseases?

SEC61B has been genetically linked to polycystic liver diseases (PLD) , and antibodies can be valuable tools for investigating disease mechanisms:

• Immunohistochemical analysis of patient samples: SEC61B antibodies can be used to examine expression patterns in liver tissue from PLD patients compared to healthy controls. Both 15087-1-AP and 51020-2-AP antibodies have been validated for IHC applications on liver tissue .

• Protein expression quantification: Western blotting with SEC61B antibodies can determine if protein levels are altered in patient-derived samples or disease models.

• Localization studies in disease models: Immunofluorescence microscopy using SEC61B antibodies can reveal mislocalization or abnormal distribution patterns in cells harboring PLD-associated mutations.

• Co-localization with cyst proteins: SEC61B antibodies can be used in dual-labeling experiments to examine relationships between the translocon and proteins known to be involved in cyst formation.

• Mechanistic studies in cellular models: After introducing PLD-associated mutations, SEC61B antibodies can help determine if:

  • ER morphology is altered

  • Protein translocation efficiency is compromised

  • Protein-protein interactions within the translocon complex are disrupted

  • Stress response pathways are abnormally activated

What considerations should be taken when interpreting SEC61B antibody staining in cancer tissue samples?

When examining SEC61B expression in cancer tissues, several important considerations should guide interpretation:

• Expression level variation: The baseline expression of SEC61B varies between tissue types. Proper controls (adjacent normal tissue from the same patient) are essential for meaningful comparison.

• Subcellular localization changes: In cancer cells, SEC61B may show altered distribution patterns within the ER or unexpected localization to other compartments. Compare with established ER markers to identify abnormal patterns.

• Tumor heterogeneity: SEC61B expression may vary within different regions of a tumor. Use tissue microarrays or multiple sections to capture this heterogeneity.

• Isotype controls and validation: For each cancer type, validate staining with appropriate isotype controls and blocking peptides to ensure specificity.

• Correlation with clinical parameters: When examining SEC61B in cancer samples, correlate staining patterns with:

  • Tumor grade and stage

  • Patient survival data

  • Response to ER stress-inducing therapies

  • Expression of UPR markers (e.g., BiP, CHOP)

• Technical considerations:

  • Antigen retrieval methods may affect staining intensity (TE buffer pH 9.0 is recommended)

  • Different SEC61B antibodies may yield varying results based on epitope accessibility

  • Polyclonal antibodies generally provide stronger staining but with potentially higher background

How should researchers reconcile contradictory results when using different SEC61B antibodies?

When faced with discrepancies between results obtained with different SEC61B antibodies, consider the following systematic approach:

• Epitope differences: Different antibodies target distinct regions of SEC61B.

  • Monoclonal antibody rAB01-4H3 recognizes amino acids 2-70 of human SEC61B .

  • Polyclonal antibodies may recognize multiple epitopes across the protein.

  • Check if protein modifications or interactions might mask specific epitopes.

• Antibody format and species differences:

  • Compare the performance of antibodies from different host species (mouse vs. rabbit).

  • Consider that IgG subclass (e.g., IgG2a for rAB01-4H3 ) may affect tissue penetration and background.

  • Assess if recombinant antibodies perform differently from hybridoma-derived ones.

• Validation experiments:

  • Perform side-by-side comparison using identical samples and protocols.

  • Include positive controls (HepG2, HeLa cells) and negative controls (SEC61B-depleted cells).

  • Conduct peptide competition assays with the immunogens used to generate each antibody.

• Technical considerations:

  • Optimize protocols for each antibody independently (concentration, incubation time, temperature).

  • Consider whether batch-to-batch variation might explain discrepancies.

  • Evaluate if differences appear in all applications or only specific techniques.

• Independent verification methods:

  • Use orthogonal techniques (mass spectrometry, RNA-seq) to resolve discrepancies.

  • Consider tagged SEC61B expression to provide an antibody-independent detection method.

What is the significance of varying molecular weight observations for SEC61B in different experimental systems?

SEC61B's calculated molecular weight is approximately 10 kDa, but observed values in experimental systems range from 10-15 kDa . This variation has biological and technical implications:

Biological factors affecting SEC61B migration:

• Post-translational modifications: SEC61B may undergo modifications (e.g., phosphorylation, ubiquitination) that alter its electrophoretic mobility.

• Splice variants: Alternative splicing could generate protein isoforms with different molecular weights.

• Species differences: Human, mouse, and rat SEC61B may show slight differences in migration patterns despite high sequence conservation.

• Protein-protein interactions: Strong interactions with SDS-resistant complexes can affect migration.

Technical factors affecting SEC61B migration:

• Gel percentage: Higher percentage gels (15-20%) provide better resolution for small proteins like SEC61B.

• Buffer systems: Tris-Tricine gels may resolve SEC61B more accurately than Tris-Glycine systems.

• Sample preparation: Heat-induced aggregation of membrane proteins can cause anomalous migration.

• Ladder calibration: Ensure molecular weight standards are appropriate for low molecular weight range.

Recommended approach:

• Include cell lysates with confirmed SEC61B expression as positive controls in each experiment.

• Use multiple antibodies targeting different epitopes to confirm band identity.

• Consider including recombinant SEC61B as a migration standard.

• Document experimental conditions that affect observed molecular weight to facilitate cross-laboratory comparisons.

How might emerging proximity labeling techniques be combined with SEC61B antibodies to advance our understanding of translocon dynamics?

Combining proximity labeling methods with SEC61B antibodies represents a powerful approach for unraveling translocon composition and dynamics:

• BioID or TurboID-SEC61B fusion proteins: Express SEC61B fused to a biotin ligase, then use SEC61B antibodies to confirm proper localization and expression level before proximity labeling experiments.

• APEX2-SEC61B for electron microscopy: Create APEX2-SEC61B fusions for EM visualization of the translocon microenvironment, validating fusion protein functionality with SEC61B antibodies.

• Split-BioID systems: Combine SEC61B with one half of a split biotin ligase and a putative interactor with the other half to detect specific interactions. SEC61B antibodies can verify expression levels of fusion constructs.

• Validation of proximity labeling results: Use SEC61B antibodies in co-IP experiments to confirm interactions identified through proximity labeling.

• Dynamic studies during ER stress: Apply SEC61B antibodies to track changes in translocon composition identified by proximity labeling under various stress conditions.

• In situ validation: Use SEC61B antibodies in combination with probes against biotinylated proteins to confirm spatial relationships between SEC61B and labeled proteins.

• Quantitative analysis of protein turnover: Combine proximity labeling with pulse-chase experiments, using SEC61B antibodies to immunoprecipitate labeled complexes at different time points.

What methodological advances could improve the specificity and sensitivity of SEC61B detection in challenging samples?

Several methodological innovations could enhance SEC61B detection in difficult samples:

• Proximity ligation assay (PLA): Combining two antibodies targeting different SEC61B epitopes in a PLA workflow could dramatically improve specificity and sensitivity in tissues with high background.

• Fluorescence-activated cell sorting (FACS): For blood or bone marrow samples, intracellular staining protocols using SEC61B antibodies could enable quantification and isolation of cells with altered SEC61B expression.

• Mass cytometry (CyTOF): Metal-conjugated SEC61B antibodies could enable highly multiplexed analysis of SEC61B expression alongside dozens of other markers in heterogeneous samples.

• Single-molecule detection methods: Techniques like stochastic optical reconstruction microscopy (STORM) combined with highly specific SEC61B antibodies could reveal nanoscale organization of translocons.

• Expansion microscopy: Physical expansion of samples before SEC61B immunostaining could improve visualization of crowded ER membranes.

• Tyramide signal amplification: For samples with low SEC61B expression, tyramide amplification could enhance detection sensitivity while maintaining spatial resolution.

• Recombinant nanobodies: Development of SEC61B-specific nanobodies could improve penetration into complex tissues and reduce background.

• Aptamer-based detection: DNA/RNA aptamers selected against SEC61B could provide alternative recognition molecules with unique advantages in certain applications.