SNAP25 Cleavage Diagram
Recombinant GST-SNAP25-EGFP is purified in order to review activation of ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ protein. Since wild type Botulinum Tox in Light Chain cleaves an C-terminal part of SNAP25, GST & EGFP are recombined with 25kDa size on each N-terminal and C-terminal, and purified in order to authentically confirm cleavage form. 75kDa GST-SNAP25-EGFP is cleaved into 50kDa and 25kDa proteins by ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’.
SNAP25 Cleavage
Size alteration after GST-SNAP25-EGFP cleavage
in-vitro SNAP25 cleavage assay
Buffer condition: 1mM DTT, 1mM HEPES, 1mM NaCl, 2uM ZnCl₂
Reaction : 37℃ 3hrs
SDS-PAGE loading condition
– 12% SDS-PAGE
– volt : 120V
in vitro SNAP25 Cleavage Assay
React GST-SNAP25-EGFR protein with various dose of ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ in vitro to see if GST-SNAP25-EGFP is cleaved by ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’.
As a result, GST-SNAP25-EGFP protein is cleaved into GST-SNAP25(50kDa) and EGFP(25kDa) depend on dose of ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’.
Cell Penetrate Assay
Permeation of Keratinocytes by ‘Methionyl r-Clostridium
Botulinum Polypeptide-1 Hexapeptide-40’
Permeation of keratinocytes
Cell treatment on Keratinocytes cell with FITC conjugated ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ protein.
After 1~6 hours, protein can be seen inside the cell. This result shows that ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ can penetrate the skin.
* Treatment condition
– HaCaT cell
– Peptide : 5μm
‘Methionyl r-Clostridium Botulinum Polypeptide-1
Hexapeptide-40′ cell penetrate test
Potential in human neuronal cell
Cell treatment on differentiated SiMa cell (Human neuroblastoma) with FITC conjugated ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ protein. After 3 hours, protein can be seen inside the cell. This result shows that ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ can be delivered to the human nerve cell, the most important target of Botulinum Protein.
* Treatment condition
– FITC conjugated : 5μg
– Time : 3hrs
– Method : confocal
‘Methionyl r-Clostridium Botulinum Polypeptide-1
Hexapeptide-40′ transdermal diffusion test
Transdermal Diffusion
Perform transdermal diffusion test by using Strat-M membrane which is a product from Millipore. Conversion of ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ amount which is penetrated from 30minutes to 24 hours, into cumulative frequency by using ELISA.
Penetrated continuously even after 24 hours.