SEC22B Antibody

What is SEC22B and what cellular functions does it regulate?

SEC22B is a vesicle trafficking protein homolog B, a SNARE family member involved in membrane fusion events. In humans, the canonical protein has 215 amino acid residues with a molecular weight of 24.7 kDa . SEC22B primarily localizes to the endoplasmic reticulum (ER) and Golgi apparatus, where it facilitates bidirectional transport between these organelles . As a v-SNARE, SEC22B interacts with cognate t-SNARE proteins such as Syntaxin 5, Bet1, and GS27 to form SNARE complexes essential for vesicular transport . SEC22B plays critical roles in:

• ER-Golgi protein transport

• Antibody secretion in plasma cells

• Phagosome maturation in immune cells

• Antigen cross-presentation by dendritic cells

SEC22B's function extends beyond basic trafficking to influence critical immune processes, making it an important target for immunological research.

What applications are most commonly validated for SEC22B antibodies?

SEC22B antibodies have been validated across multiple applications with varying sensitivity and specificity profiles:

For optimal results, each application requires specific sample preparation protocols. For example, in immunohistochemistry, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has shown superior results for detecting SEC22B in paraffin-embedded tissue sections .

What species reactivity is available for SEC22B antibodies?

Most commercially available SEC22B antibodies demonstrate cross-reactivity with multiple species due to the high conservation of this protein:

When planning cross-species experiments, researchers should verify the specific epitope recognized by their antibody of choice, as certain regions may show lower conservation across species.

Advanced Experimental Considerations

SEC22B has been identified as a critical regulator of plasma cell maintenance and antibody secretion . When designing experiments to study this function:

• Secretion rate measurement: Implement droplet microfluidic-based techniques to assess antibody secretion at the single-cell level. This approach can detect differential secretion rates between wild-type and SEC22B-deficient plasma cells (e.g., wild-type cells secreted ~12 IgM/s on day 2 versus ~2-4 IgM/s in SEC22B-deficient cells) .

• Transcriptional profiling: Monitor plasma cell maturation markers in SEC22B-deficient models:

  • Measure expression of plasma cell markers (CD93, Tnfrsf13b)

  • Assess plasma cell master regulators (Prdm1, Xbp1, Irf4)

  • Evaluate B cell regulators (Pax5, Bach2)

• Antibody titer measurement: Quantify multiple antibody isotypes (IgM, IgG1, IgA, IgG3) in serum to comprehensively assess the impact of SEC22B deficiency:

  Antibody Isotype	Reduction in SEC22B B-KO mice	Technical Considerations
  Igκ	~40-fold	Represents ~90% of secreted antibodies in mice
  IgG1	~55-fold	Most dramatically affected isotype
  IgM	~40-fold	Early responder in immune reactions
  IgA	~30-fold	Important for mucosal immunity
  IgG3	~20-fold	Least affected among tested isotypes

• Plasma cell quantification: Analyze both frequency and absolute numbers of plasma cells in relevant tissues (spleen, bone marrow) using flow cytometry, with particular attention to mature CD19-B220- plasma cell populations .

How should researchers experimentally approach the study of SEC22B in antigen cross-presentation?

The role of SEC22B in antigen cross-presentation has been controversial, with contradictory findings between different experimental approaches . Consider these methodological approaches:

• In vitro cross-presentation assays:

  • Use multiple antigen forms (soluble, particulate) with OVA as a model antigen

  • Include both short-term and long-term incubation protocols

  • Implement rigorous controls including direct peptide presentation (SIINFEKL)

  • Employ multiple readouts: T cell activation (CD69, CD25), proliferation (CFSE dilution), and cytokine production

• Flow cytometry detection:

  • Implement careful gating strategies to exclude non-specific signals

  • For analysis of specific CD8+ T cells, use a fluorescent multimer approach (e.g., MHC class I tetramers loaded with specific epitopes)

  • Include a DUMP channel to exclude dead cells, B lymphocytes, and myeloid cells

  • When analyzing T cell populations, use CD3 positivity before differentiating CD4+ from CD8+ cells

• In vivo cross-presentation models:

  • Employ tumor models to assess SEC22B's role in anti-tumor immunity

  • Evaluate responses to checkpoint inhibitors (e.g., anti-PD-1) in SEC22B-deficient models

  • Consider cell-associated antigen models, which appear particularly dependent on SEC22B

• Technical considerations:

  • Exclude potential endotoxin contamination in antigen preparations

  • Ensure beads are properly excluded by their scatter plot distribution in flow cytometry

  • Use endotoxin-free OVA from reliable suppliers to prevent confounding results

What mechanisms underlie SEC22B's involvement in phagosome maturation?

SEC22B plays a crucial role in phagosome maturation through tethering endoplasmic reticulum-phagosome membrane contact sites (MCS), independent of the known tether STIM1 . For researchers investigating this function:

• Phospholipid dynamics measurement:

  • Monitor multiple phagosomal phospholipids (PI(3)P, PS, PI(4)P) in wild-type vs. SEC22B knockdown cells

  • Implement rescue experiments with:

    • Wild-type SEC22B expression

    • The artificial tether MAPPER

    • The MCS-disrupting mutant SEC22B-P33

• Protein interaction studies:

  • Investigate SEC22B co-precipitation with lipid exchange proteins (e.g., ORP8, a PS/PI(4)P exchange protein)

  • Compare effects of wild-type vs. mutant ORP8 on phagosomal PI(4)P levels and antigen degradation

  • Examine interactions with other SNARE proteins (e.g., Syntaxin 4)

• Calcium signaling analysis:

  • Measure calcium flux in SEC22B-deficient cells, as SEC22B knockdown has been shown to increase calcium signaling

  • Correlate calcium signaling with phagolysosome fusion and antigen degradation rates

This multifaceted approach can help distinguish between SEC22B's fusion-dependent and tethering-dependent functions in phagosome biology.

What are the critical parameters for optimizing SEC22B antibody use in Western blotting?

Western blotting is one of the most widely validated applications for SEC22B antibodies. For optimal results:

• Sample preparation:

  • NETN buffer has been validated for effective lysate preparation

  • Include protease inhibitors to prevent degradation

  • Denature samples at 95°C for 5 minutes in standard Laemmli buffer

• Gel selection and transfer:

  • Use 12-15% polyacrylamide gels to effectively resolve the 24-25 kDa SEC22B protein

  • Semi-dry transfer systems work well with standard PVDF membranes

• Antibody selection and dilution:

  • Monoclonal antibodies (e.g., 29-F7) provide consistent results at 1:2000-1:12000 dilutions

  • Polyclonal antibodies may require higher concentrations (1:1000) but can offer advantages in detecting modified forms of the protein

  • Recombinant monoclonal antibodies (e.g., EPR12335) offer superior batch-to-batch consistency

• Signal detection:

  • ECL detection systems have been validated for SEC22B visualization

  • Expected band size is approximately 24-25 kDa

  • Validate specificity using knockout/knockdown controls when possible

How should researchers approach immunohistochemical detection of SEC22B?

For effective immunohistochemical detection of SEC22B in tissue sections:

• Antigen retrieval optimization:

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has shown superior results compared to citrate buffer (pH 6.0)

  • 10-20 minute retrieval times at near-boiling temperatures typically yield optimal results

• Blocking and antibody incubation:

  • 10% goat serum has been validated for effective blocking

  • Antibody concentrations of 2 μg/ml with overnight incubation at 4°C provide optimal staining

  • Secondary antibody incubation for 30 minutes at 37°C helps minimize background

• Detection systems:

  • HRP-conjugated secondary antibodies with DAB chromogen provide good contrast

  • For fluorescence, DyLight 488/Cy3-conjugated secondary antibodies offer excellent sensitivity

• Tissue-specific considerations:

  • SEC22B detection has been validated in multiple human tissues including colorectal adenocarcinoma, ovarian serous adenocarcinoma, and placenta

  • When comparing normal vs. pathological tissues, examine both staining intensity and subcellular distribution patterns

• Controls:

  • Include isotype controls to assess non-specific binding

  • When possible, include tissue from SEC22B knockout models as negative controls

What considerations are important when using SEC22B antibodies for co-localization studies?

Co-localization studies are crucial for understanding SEC22B's dynamic interactions with other proteins. To optimize these experiments:

• Fixation and permeabilization:

  • 4% paraformaldehyde fixation preserves both SEC22B and organelle structure

  • 0.1% Triton X-100 permeabilization allows antibody access while preserving membrane structures

• Co-staining partners:

  • ER markers (e.g., calnexin, PDI) to assess ER localization

  • Golgi markers (e.g., GM130, TGN46) to evaluate Golgi association

  • ERGIC markers to assess intermediate compartment localization

  • Organelle-specific markers for specialized studies (e.g., phagosome, mitochondria)

• Imaging considerations:

  • Confocal microscopy is essential for accurate co-localization assessment

  • Super-resolution techniques (STED, STORM) can provide enhanced detail of SEC22B distribution at contact sites

  • Z-stack acquisition ensures complete spatial assessment of co-localization

• Quantitative analysis:

  • Use Pearson's correlation coefficient or Manders' overlap coefficient for quantitative co-localization assessment

  • Analyze multiple cells across independent experiments for statistical validity

  • Consider live-cell imaging with fluorescently tagged SEC22B to assess dynamic interactions

How can researchers address conflicting results between different SEC22B antibody clones?

Discrepancies between different SEC22B antibodies may arise due to epitope specificity, conformation sensitivity, or cross-reactivity. To address this:

• Epitope mapping:

  • Compare the immunogen sequences used to generate each antibody

  • Antibodies targeting amino acids 100-150 of human SEC22B have shown good specificity

  • Consider using antibodies recognizing different epitopes to confirm results

• Validation approach:

  • Implement siRNA/shRNA knockdown to validate specificity

  • When possible, use tissues/cells from SEC22B knockout models

  • Perform blocking peptide competition assays to confirm specificity

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with SEC22 paralogs (SEC22A, SEC22C)

  • Validate species cross-reactivity when working with non-human models

  • Consider potential post-translational modifications that might affect epitope recognition

What experimental approaches help resolve contradictions in SEC22B functional studies?

The literature contains notable contradictions regarding SEC22B's role in processes like antigen cross-presentation . To address such discrepancies:

• Comprehensive experimental design:

  • Test multiple cellular contexts (cell lines vs. primary cells)

  • Compare different gene silencing approaches (siRNA, shRNA, CRISPR/Cas9)

  • Implement rescue experiments with wild-type and mutant SEC22B

• Technical validation:

  • Verify knockout/knockdown efficiency at both protein and mRNA levels

  • Check for compensatory expression of related genes (SEC22A, SEC22C)

  • Perform RNA-seq to detect potential off-target effects of silencing approaches

• Functional redundancy analysis:

  • Test models with combined knockdown of potential compensatory proteins

  • Use domain-specific mutants to dissect specific functional aspects

  • Consider cell-type specific differences in SEC22B dependency

• Physiological relevance assessment:

  • Compare in vitro findings with in vivo models

  • Evaluate effects under both steady-state and stimulated conditions

  • Consider developmental stage and differentiation status dependencies

By implementing these approaches systematically, researchers can better understand the true functional significance of SEC22B in their specific experimental system.

What are promising approaches for studying SEC22B's role in ER and mitochondrial homeostasis?

Recent findings link SEC22B to the regulation of endoplasmic reticulum and mitochondrial structure, particularly in plasma cells . To investigate this emerging function:

• Organelle morphology assessment:

  • Implement high-resolution microscopy techniques (electron microscopy, super-resolution) to visualize ER and mitochondrial architecture

  • Quantify morphological parameters (network connectivity, tubule length, cristae structure)

  • Analyze dynamic changes using live-cell imaging with organelle-specific markers

• Functional assays:

  • Assess mitochondrial membrane potential using JC-1 or TMRE dyes

  • Measure oxygen consumption rate and extracellular acidification rate

  • Evaluate calcium homeostasis in both ER and mitochondria using organelle-targeted calcium indicators

• Protein-protein interaction studies:

  • Identify SEC22B interaction partners at ER-mitochondria contact sites

  • Investigate SEC22B's relationship with known ER-mitochondria tethering complexes (ERMES, ERMCS)

  • Use proximity labeling techniques (BioID, APEX) to identify novel interaction partners

• Stress response analysis:

  • Examine unfolded protein response activation in SEC22B-deficient cells

  • Assess mitochondrial stress responses and quality control mechanisms

  • Investigate susceptibility to ER and mitochondrial stressors (tunicamycin, thapsigargin, CCCP)

What novel therapeutic applications might emerge from SEC22B research?

SEC22B's critical roles in immune function suggest potential therapeutic implications:

• Targeting plasma cell disorders:

  • SEC22B inhibition may represent a novel approach for targeting pathological plasma cells in multiple myeloma or autoimmune diseases

  • Development of small molecule inhibitors or degraders targeting SEC22B

  • Assessment of SEC22B expression as a prognostic marker in plasma cell dyscrasias

• Cancer immunotherapy enhancement:

  • SEC22B modulation might enhance antigen cross-presentation for improved cancer vaccine efficacy

  • SEC22B-dependent pathways appear critical for anti-PD-1 immunotherapy responses

  • Engineering dendritic cells with optimized SEC22B expression could enhance cancer immunotherapy approaches

• Autoimmune disease intervention:

  • Targeting SEC22B may represent a novel approach to modulate antibody production in antibody-mediated autoimmune diseases

  • SEC22B inhibition could potentially reduce pathogenic antibody titers while preserving other immune functions

• Biomarker development:

  • Assessment of SEC22B expression or modification patterns as biomarkers for disease progression or treatment response

  • Development of SEC22B autoantibody detection for potential autoimmune conditions