SEC10a Antibody

Here’s a structured, research-focused FAQ for SEC10 (EXOC5) antibodies, synthesized from peer-reviewed methodologies and empirical data:

Advanced Data Interpretation

Q3: How to resolve discrepancies in SEC10 expression across cell lines?

Solution: Normalize using total protein quantification (e.g., Stain-Free gels) and correlate with functional assays (e.g., vesicle trafficking assays).

Q4: How to optimize SEC10 detection in low-abundance samples?

• Signal enhancement:

  • Use chemiluminescent substrates with high dynamic range.

  • Increase primary antibody concentration (e.g., 1–2 µg/mL for ab241472 ).

  • Employ signal amplification systems (e.g., biotin-streptavidin).

Technical Troubleshooting

Q5: How to address nonspecific bands in SEC10 Western blots?

• Causes and fixes:

  • Proteolytic degradation: Add fresh protease inhibitors during lysis.

  • Cross-reactivity: Pre-adsorb antibody with lysate from SEC10-knockout cells.

  • Overexposure: Reduce secondary antibody concentration (e.g., 1:10,000 dilution ).

Q6: Can SEC10 antibodies be used in multiplex assays with other exocyst components?

• Strategy:

  • Use species-specific primary antibodies (e.g., mouse anti-SEC8 + rabbit anti-SEC10).

  • Pair with spectrally distinct fluorescent secondaries (e.g., Alexa Fluor 488/647).

  • Validate absence of cross-reactivity via single-antibody controls .

Functional Research Applications

Q7: How to design co-IP experiments to study SEC10-protein interactions?

• Protocol:

  • Lyse cells in NETN buffer to preserve weak interactions .

  • Use 1–2 mg lysate and 6 µg SEC10 antibody per IP reaction .

  • Elute with low-pH buffer (e.g., 0.1 M glycine, pH 2.5) to minimize antibody contamination.

Q8: What methods confirm SEC10’s role in exocytosis?

• Approaches:

  • siRNA-mediated SEC10 knockdown + cargo secretion assays (e.g., β-hexosaminidase release).

  • Co-localization studies with fluorescently labeled vesicles (e.g., pH-sensitive GFP-Rab11).

Cross-Species and Isoform Considerations

Q9: Does ab241472 recognize murine SEC10 isoforms?

• Data:

  • Detects 82 kDa band in TCMK-1 (mouse kidney) and NIH/3T3 lysates .

  • No cross-reactivity with non-mammalian orthologs confirmed.

Q10: How to validate SEC10 antibody specificity for splice variants?

• Steps:

  • Compare lysates from cells overexpressing known isoforms (e.g., GRINL1A vs. GRINL1A-4/5 ).

  • Use isoform-specific knockout models or blocking peptides.