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TEST RESULTS
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 Toxin 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
GST
GST
SNAP25
SNAP25
EGFP
EGFP
75kDa
50kDa + 25kDa
in-vitro SNAP25 cleavage assay
GST-SNAP25-EGFP
GST-SNAP25
EGFP
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
Vehicle
Lc
BPMed_BoLCA
1 hr
3 hr
6 hr
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 skin.
* Treatment condition
- HaCaT cell
- Peptide : 5μm
Vehicle
Lc
BPMed_BoLCA
'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 delevered 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.