SEC23B Antibody

What is SEC23B and what are its primary cellular functions?

SEC23B is a component of coat protein complex II (COPII), which canonically transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. It functions as a GTPase-activating protein that activates the SAR1 GTPase and plays a critical role in cargo recognition during protein trafficking. Beyond this classical function, wild-type SEC23B has recently been discovered to have non-canonical roles, particularly within cellular stress response pathways. The protein can localize to the nucleus in addition to its classical distribution at the ER/Golgi interface and interacts with nuclear proteins and those involved in ER stress, protein ubiquitination, and EIF2 signaling pathways . SEC23B levels increase in response to ER stress, supporting its role as a cellular stress response sensor and/or effector .

What disease associations have been identified with SEC23B mutations?

SEC23B mutations have significant clinical implications across different mutation types. Germline recessive loss-of-function mutations in SEC23B cause congenital dyserythropoietic anemia type II (CDAII), the most common form of congenital dyserythropoietic anemia . Heterozygous change-of-function mutations in SEC23B result in increased predisposition to cancer . The severity of CDAII phenotypes correlates with mutation types - compound heterozygosity for a missense and a nonsense mutation typically produces more severe clinical presentation, lower reticulocyte count, higher serum ferritin level, and sometimes more pronounced transfusion needs compared to homozygosity or compound heterozygosity for two missense mutations . Notably, hypomorphic mutations of the SEC23B gene can account for milder clinical phenotypes .

What are the molecular weight and structural characteristics of SEC23B protein?

SEC23B has a calculated molecular weight of 86 kDa, though it is typically observed at 83-86 kDa range in Western blot applications . The protein contains multiple putative nuclear localization and export signals that regulate nuclear-cytoplasmic transport, which explains its ability to localize to both the nucleus and the ER/Golgi interface . SEC23B is encoded by the SEC23B gene, with the full protein name being "Sec23 homolog B (S. cerevisiae)." Its UniProt ID is Q15437, and the GenBank accession number is BC005404 .

What criteria should researchers consider when selecting a SEC23B antibody?

When selecting a SEC23B antibody for research applications, researchers should evaluate several key parameters: (1) Validated reactivity with the species of interest (e.g., human, mouse); (2) Confirmed specificity through proper controls including knockout/knockdown validation; (3) Validated applications such as Western blot, immunoprecipitation, or immunofluorescence that align with experimental needs; (4) Recognition of relevant epitopes, especially when studying specific mutations or isoforms; and (5) Clone type (monoclonal vs. polyclonal) based on experimental requirements. For human samples, antibodies like the polyclonal 32278-1-AP have demonstrated reactivity in applications such as Western blot and ELISA . Researchers should review validation data and literature citations to ensure the antibody performs reliably in their specific experimental context.

How should SEC23B antibodies be validated for specificity in experimental settings?

Rigorous validation of SEC23B antibodies should include multiple complementary approaches: (1) Western blot analysis comparing SEC23B-expressing cell lines (e.g., HeLa, HepG2, K-562, Raji cells) with SEC23B-knockout or knockdown models; (2) Immunoprecipitation followed by mass spectrometry to confirm target pull-down; (3) Peptide competition assays to demonstrate binding specificity; (4) Cross-reactivity assessment with the paralog SEC23A, which shares structural similarities; (5) Immunofluorescence co-localization studies comparing antibody staining patterns with known SEC23B distribution at the ER/Golgi interface and, under certain conditions, in the nucleus and nucleoli . For genetic research, validation should include testing across samples with different SEC23B mutations to confirm detection capabilities for variant forms.

What are the optimal storage and handling conditions for SEC23B antibodies?

SEC23B antibodies require careful handling to maintain their functionality. For example, antibodies like the 32278-1-AP should be stored at -20°C in appropriate buffer conditions (PBS with 0.02% sodium azide and 50% glycerol at pH 7.3) . Long-term stability is typically one year after shipment when properly stored. While some antibody preparations (particularly those at higher concentrations) may not require aliquoting for -20°C storage, for most research applications, it is advisable to create single-use aliquots to avoid freeze-thaw cycles that can degrade antibody performance. Working dilutions should be prepared fresh and used within 24 hours. Some formulations may contain BSA (0.1%) as a stabilizer . Researchers should always consult product-specific protocols and avoid contamination during handling.

What are the recommended protocols for using SEC23B antibodies in Western blot applications?

For optimal Western blot results with SEC23B antibodies, researchers should follow these methodological guidelines: (1) Sample preparation: Lyse cells in RIPA buffer supplemented with protease inhibitors, ensuring complete lysis of nuclear and ER membranes where SEC23B localizes; (2) Protein loading: Load 20-40 μg of total protein per lane; (3) SDS-PAGE: Use 8-10% gels for optimal resolution of the 83-86 kDa SEC23B protein; (4) Transfer: Employ wet transfer systems for efficient transfer of higher molecular weight proteins; (5) Blocking: Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature; (6) Primary antibody incubation: Dilute SEC23B antibodies at 1:1000-1:4000 in blocking buffer and incubate overnight at 4°C ; (7) Detection: Use appropriate secondary antibodies and detection systems compatible with your imaging equipment. Always include positive controls (HeLa, HepG2, K-562, or Raji cell lysates) and, when possible, negative controls (SEC23B-knockout samples) .

How can researchers effectively study SEC23B cellular localization under different stress conditions?

To study SEC23B cellular localization under various stress conditions, researchers should employ immunofluorescence microscopy with these methodological considerations: (1) Cell culture: Grow cells on poly-L-lysine coated coverslips and induce relevant stress conditions (e.g., proteasome inhibition with MG132, ER stress with tunicamycin or thapsigargin); (2) Fixation: Use 4% paraformaldehyde for 15 minutes followed by permeabilization with 0.1% Triton X-100; (3) Immunostaining: Incubate with validated SEC23B antibodies and co-stain with markers for relevant compartments (e.g., Calnexin for ER, GM130 for Golgi, fibrillarin for nucleoli); (4) Confocal microscopy: Utilize Z-stack imaging to precisely determine subcellular localization; (5) Quantification: Perform quantitative analysis of SEC23B distribution across cellular compartments under different stress conditions . This approach has revealed that wild-type SEC23B can localize to cell nucleoli under proteasome inhibition conditions, with distribution patterns distinct from those observed in cells expressing mutant SEC23B .

What techniques are recommended for studying SEC23B interactions with other proteins?

For comprehensive analysis of SEC23B protein interactions, researchers should employ multiple complementary techniques: (1) Co-immunoprecipitation: Use SEC23B antibodies to pull down protein complexes from cell lysates, followed by immunoblotting for suspected interacting partners; (2) Proximity ligation assay (PLA): Visualize protein-protein interactions in situ with spatial resolution; (3) Mass spectrometry: Perform unbiased proteomic analysis of SEC23B immunoprecipitates to identify novel interaction partners; (4) Yeast two-hybrid or mammalian two-hybrid assays: Validate direct protein-protein interactions; (5) FRET or BRET analysis: Measure real-time interactions in live cells; (6) SEC23B-UBA52 interaction validation: Use co-immunoprecipitation followed by Western blot to specifically examine this important interaction . These approaches have revealed that wild-type SEC23B interacts with nuclear proteins and central proteins in the ER stress, protein ubiquitination, and EIF2 signaling pathways .

How do animal models of SEC23B deficiency differ from human disease phenotypes?

A significant species-specific difference exists in SEC23B deficiency phenotypes. While germline recessive loss-of-function mutations in human SEC23B cause congenital dyserythropoietic anemia type II (CDAII), mice completely deficient for SEC23B do not exhibit anemia but instead die shortly after birth with degeneration of professional secretory tissues, particularly the pancreas . In SEC23B-deficient embryonic mouse pancreas, both exocrine and endocrine tissues show defects shortly after differentiation - pancreatic acini are completely devoid of zymogen granules, and the ER is severely distended . This species-specific difference has been attributed to the expression of the SEC23A paralog, as expression of full SEC23A protein from the endogenous regulatory elements of Sec23b completely rescues the SEC23B-deficient mouse phenotype . These interspecies differences highlight important considerations for translational research using mouse models.

What experimental approaches can be used to study SEC23B mutations associated with congenital dyserythropoietic anemia?

To study SEC23B mutations associated with CDAII, researchers should consider these methodological approaches: (1) Patient-derived lymphoblastoid cell lines harboring either wild-type or mutant SEC23B for comparative functional studies ; (2) CRISPR/Cas9-mediated introduction of specific SEC23B mutations into erythroid cell lines like HUDEP-2, which upon differentiation exhibit CDAII features ; (3) Genotype-phenotype correlation studies using patient samples with varying mutation types (missense vs. nonsense) to assess disease severity markers ; (4) Rescue experiments through increased expression of SEC23A in SEC23B-deficient cells to evaluate potential therapeutic strategies ; (5) Erythroid-specific conditional knockout mouse models targeting both Sec23a and Sec23b to recapitulate CDAII features, as mice with erythroid-specific deletion of all four Sec23 alleles die in mid-embryogenesis with CDAII-like features . These approaches collectively provide insights into disease mechanisms and potential therapeutic interventions.

How can researchers differentiate between canonical and non-canonical functions of SEC23B in experimental settings?

To distinguish between canonical COPII-dependent and non-canonical functions of SEC23B, researchers should implement these specialized experimental approaches: (1) Subcellular localization studies comparing SEC23B distribution with other COPII components under normal and stress conditions; (2) Mutation analysis focusing on specific domains required for COPII assembly versus domains involved in other cellular functions; (3) Genetic manipulation studies selectively disrupting SEC23B's interaction with other COPII components while preserving other functions; (4) Proteomic analysis comparing SEC23B interactome under conditions that favor or disfavor COPII assembly; (5) Functional assays measuring conventional protein trafficking from ER to Golgi versus stress response pathways . These strategies have revealed that wild-type SEC23B can localize to cell nucleoli independently of COPII under proteasome inhibition conditions and interacts with proteins involved in ER stress response, demonstrating its role beyond canonical protein trafficking .

What are the methodological approaches for studying SEC23B-mediated cellular stress responses?

To investigate SEC23B's role in cellular stress responses, researchers should employ these systematic approaches: (1) Stress induction experiments using various stressors (ER stress inducers like tunicamycin, thapsigargin; proteasome inhibitors like MG132; oxidative stress inducers) while monitoring SEC23B levels, localization, and post-translational modifications; (2) Time-course analysis of SEC23B expression and localization following stress induction; (3) Chromatin immunoprecipitation (ChIP) assays to identify potential direct involvement in transcriptional regulation during stress; (4) SEC23B knockout/knockdown followed by transcriptomic and proteomic analysis to identify affected stress response pathways; (5) Rescue experiments with wild-type versus mutant SEC23B to identify critical domains for stress response functions . These approaches have demonstrated that SEC23B levels increase in response to ER stress in patient-derived lymphoblastoid cell lines harboring either wild-type or mutant SEC23B, supporting its role as a cellular stress response sensor and/or effector .

What are the technical challenges in differentiating between SEC23A and SEC23B in experimental systems?

Differentiating between the paralogs SEC23A and SEC23B presents several technical challenges requiring specific methodological solutions: (1) Antibody specificity validation through side-by-side testing on SEC23A- and SEC23B-knockout samples; (2) Peptide mapping to identify unique epitopes for generating paralog-specific antibodies; (3) RNA interference approaches using siRNAs designed against non-conserved regions; (4) CRISPR/Cas9 targeting of paralog-specific genomic regions; (5) Rescue experiments evaluating functional redundancy, as expression of SEC23A from SEC23B regulatory elements can rescue SEC23B deficiency in mice ; (6) Quantitative real-time PCR with paralog-specific primers targeting non-conserved regions; (7) Mass spectrometry identification of unique peptides for each paralog. These approaches are essential for accurate interpretation of experimental results, particularly given the functional overlap between these paralogs in certain cellular contexts .

What are the current hypotheses explaining species-specific differences in SEC23B deficiency phenotypes?

The striking difference between human CDAII and mouse pancreatic failure phenotypes due to SEC23B deficiency has generated several testable hypotheses: (1) Differential expression patterns of SEC23A and SEC23B across species and tissues may explain tissue-specific vulnerability; (2) Species-specific regulatory elements controlling SEC23A expression may allow compensation in certain tissues in mice but not humans; (3) Evolutionary divergence in protein interfaces between SEC23B and its interacting partners may result in different functional dependencies across species; (4) Differences in erythropoiesis between humans and mice may alter requirements for SEC23B; (5) The presence of species-specific modifier genes may influence phenotypic outcomes . Research supporting these hypotheses includes the observation that expression of SEC23A from SEC23B regulatory elements rescues SEC23B-deficient mice, and that mice with erythroid-specific deletion of all four Sec23 alleles exhibit CDAII-like features, suggesting compensation by SEC23A in mouse erythroid cells .

Table 1: Comparison of SEC23B Mutation Types and Associated Clinical Phenotypes

Table 2: SEC23B Antibody Applications and Recommended Protocols