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Chemistry

Botulinum nörotoksin Işık Zinciri Modülatörlerin Belirlenmesi ve Karakterizasyonu için bir Yüksek verim uyumlu FRET tabanlı Platformu

doi: 10.3791/50908 Published: December 27, 2013

Materials

Name Company Catalog Number Comments
HEPES Teknova H1021
Tween-20 Fisher Scientific BP337-100
Methanol (HPLC-grade) Sigma-Aldrich 34860
Isopropanol (HPLC-grade) Sigma-Aldrich 650447
96-well Black assay plate Costar 3915
384-well Low-volume black assay plate Greiner 788076
SNAPtide FITC/Dabcyl substrate List Biological Laboratories 521 FRET-based BoNT/A LC substrate
Pin cleaning solution V&P Scientific VP 110
Lint-free blotting paper V&P Scientific VP 540DB
Biomek Seal and Sample Aluminum foil lids Beckman Coulter 538619

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References

  1. Schantz, E. J., Johnson, E. A. Properties and use of botulinum toxin and other microbial neurotoxins in medicine. Microbiol. Rev. 56, 80-99 (1992).
  2. Sloop, R. R., Cole, B. A., Escutin, R. O. Human response to botulinum toxin injection: type B compared with type A. Neurology. 49, 189-194 (1997).
  3. Willis, B., Eubanks, L. M., Dickerson, T. J., Janda, K. D. The strange case of the botulinum neurotoxin: using chemistry and biology to modulate the most deadly poison. Angew. Chem. Int. Ed. Engl. 47, 8360-8379 (2008).
  4. Tacket, C. O., Shandera, W. X., Mann, J. M., Hargrett, N. T., Blake, P. A. Equine antitoxin use and other factors that predict outcome in type A foodborne botulism. Am. J. Med. 76, 794-798 (1984).
  5. Capek, P., et al. Enhancing the Pharmacokinetic Properties of Botulinum Neurotoxin Serotype A Protease Inhibitors Through Rational Design. ACS Chem. Neurosci. 2, 288-293 (2011).
  6. Schmidt, J. J., Bostian, K. A. Proteolysis of synthetic peptides by type A botulinum neurotoxin. J. Protein Chem. 14, 703-708 (1995).
  7. Schmidt, J. J., Bostian, K. A. Endoproteinase activity of type A botulinum neurotoxin: substrate requirements and activation by serum albumin. J. Protein Chem. 16, 19-26 (1997).
  8. Schmidt, J. J., Stafford, R. G., Bostian, K. A. Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite. FEBS Lett. 435, 61-64 (1998).
  9. Schmidt, J. J., Stafford, R. G., Millard, C. B. High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F. Anal. Biochem.. 296, 130-137 (2001).
  10. Schmidt, J. J., Stafford, R. G. Fluorigenic substrates for the protease activities of botulinum neurotoxins serotypes A, B, and F. Appl. Environ. Microbiol. 69, 297-303 (2003).
  11. Pellett, S. Progress in cell based assays for botulinum neurotoxin detection. Curr. Top. Microbiol. Immunol. 364, 257-285 (2013).
  12. Boldt, G. E., et al. Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin a inhibitors. J. Comb. Chem. 8, 513-521 (2006).
  13. Henkel, J. S., et al. Catalytic properties of botulinum neurotoxin subtypes A3 and A4. Biochemistry. 48, 2522-2528 (2009).
  14. Joshi, S. G. Detection of biologically active botulinum neurotoxin--A in serum using high-throughput FRET-assay. J. Pharmacol. Toxicol. Methods. 65, 8-12 (2012).
  15. Smith, G. R., et al. Reexamining hydroxamate inhibitors of botulinum neurotoxin serotype A: extending towards the beta-exosite. Bioorg. Med. Chem. Lett. 22, 3754-3757 (2012).
  16. Eubanks, L. M., et al. An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists. Proc. Natl. Acad. Sci. U.S.A. 104, 2602-2607 (2007).
  17. Baldwin, M. R., Bradshaw, M., Johnson, E. A., Barbieri, J. T. The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability. Protein Expr. Purif. 37, 187-195 (2004).
  18. Zhang, J. H., Chung, T. D., Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screen. 4, 67-73 (1999).
  19. Capkova, K., Hixon, M. S., McAllister, L. A., Janda, K. D. Toward the discovery of potent inhibitors of botulinum neurotoxin A: development of a robust LC MS based assay operational from low to subnanomolar enzyme concentrations. Chem. Commun. 3525-3527 (2008).
  20. Pires-Alves, M., Ho, M., Aberle, K. K., Janda, K. D., Wilson, B. A. Tandem fluorescent proteins as enhanced FRET-based substrates for botulinum neurotoxin activity. Toxicon. 53, 392-399 (2009).
Botulinum nörotoksin Işık Zinciri Modülatörlerin Belirlenmesi ve Karakterizasyonu için bir Yüksek verim uyumlu FRET tabanlı Platformu
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Cite this Article

Caglič, D., Bompiani, K. M., Krutein, M. C., Čapek, P., Dickerson, T. J. A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators. J. Vis. Exp. (82), e50908, doi:10.3791/50908 (2013).More

Caglič, D., Bompiani, K. M., Krutein, M. C., Čapek, P., Dickerson, T. J. A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators. J. Vis. Exp. (82), e50908, doi:10.3791/50908 (2013).

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