SEC13B Antibody

Here’s a structured FAQ for SEC13B antibody research, synthesized from peer-reviewed studies and technical documentation:

How to validate SEC13B antibody specificity in experimental systems?

Methodology:

• Perform siRNA-mediated knockdown of SEC13B in target cell lines (e.g., HEK293, HeLa) and compare Western blot signals pre-/post-knockdown .

• Use recombinant SEC13B protein (e.g., E. coli-derived SEC13B, residues Gly123-Gln322) for competitive inhibition assays to confirm antibody binding specificity .

• Cross-validate with mass spectrometry after immunoprecipitation to identify co-purified proteins (e.g., SUF4 in Arabidopsis) .

Key Data:

What experimental conditions optimize SEC13B detection in immunofluorescence?

Protocol:

• Fix cells with 4% paraformaldehyde (15 min, RT) and permeabilize with 0.1% Triton X-100.

• Use 0.3 µg/mL SEC13B antibody (validated in HeLa cells) with 3-hour incubation .

• Counterstain with DAPI and validate localization against known markers (e.g., nuclear pore complex proteins) .

Critical Controls:

• Include SEC13B-deficient cell lines (e.g., CRISPR-edited) to confirm signal absence.

• Compare cytoplasmic vs. nuclear signals under ER stress (e.g., tunicamycin treatment) to assess COPII-related localization .

How to resolve contradictions in SEC13B interaction data across studies?

Case Analysis:

• Conflict: SEC13B binds SUF4 in Arabidopsis but not PpSec13b in Phytophthora .

  • Solution: Test ortholog-specific interactions using pull-down assays with truncated SUF4 domains (e.g., Pro-rich domain, residues 101–367) .

Data Interpretation:

How to differentiate SEC13B functional roles in COPII vs. nuclear pore complexes?

Strategies:

• Knockdown + Rescue: Silence SEC13B and reintroduce mutants (e.g., ∆NPC-binding motif) to assess ER-Golgi trafficking (COPII) vs. nuclear mRNA export .

• Proximity Ligation Assay (PLA): Use antibodies against SEC13B and Sec31A (COPII) or Nup98 (NPC) to quantify spatial associations .

Critical Findings:

• SEC13B depletion disrupts COPII vesicle formation (↓VSVG-GFP secretion) but not nuclear pore integrity (retained RanGAP1 localization) .

What methodological adjustments improve SEC13B detection in co-immunoprecipitation?

Optimization Steps:

• Lysis Buffer: Use 1% digitonin (vs. NP-40) to preserve weak interactions (e.g., SEC13B-SUF4) .

• Crosslinker: Apply DSS (2 mM, 30 min) to stabilize transient interactions before immunoprecipitation .

• Elution: Compete with 0.2 mg/mL SEC13B peptide (residues 200–250) to minimize antibody leaching .

Troubleshooting Table:

Technical Notes

• Antibody Clones: Rabbit monoclonal MAB9055 (R&D Systems) shows consistent reactivity in reducing SDS-PAGE .

• Contradictory Localization: Nuclear vs. cytoplasmic signals may reflect cell cycle-dependent SEC13B redistribution (validate via synchronized cells) .