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567333
Sigma-AldrichSNAPtide® Botulinum Toxin A Substrate, Fluorogenic - Calbiochem
SNAPtide® Botulinum Toxin A Substrate, Fluorogenic, is a synthetic peptide that acts as a FRET substrate with the fluorophore o-aminobenzoic acid and the acceptor chromophore 2,4-dinitrophenol (DNP).
More>>SNAPtide® Botulinum Toxin A Substrate, Fluorogenic, is a synthetic peptide that acts as a FRET substrate with the fluorophore o-aminobenzoic acid and the acceptor chromophore 2,4-dinitrophenol (DNP). Less<<
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Description
Overview
A synthetic peptide, fluorescence resonance energy transfer (FRET) substrate containing the N-terminally-linked fluorophore o-aminobenzoic acid (Abz) and the acceptor chromophore 2,4-dinitrophenol (DNP) linked to the C-terminus. The substrate is based on the native botulinum toxin type A cleavage site found in the synaptosome-associated protein SNAP-25. Cleavage of the substrate by botulinum toxin releases the fluorophore and full fluorescence is restored.
Catalogue Number
567333
Brand Family
Calbiochem®
References
References
Bigalke, H. and Shoer, L.F. 2000. In: Handbook of Experimental Pharmacology,145, Bacterial Protein Toxins (K. Aktories and I. Just, Eds.) pp. 407-443, Springer-Verlag, Berlin. Schiavo, G., et al. 1993. J. Biol. Chem.268, 23784.
SNAPtide® Botulinum Toxin A Substrate, Fluorogenic - Calbiochem Certificates of Analysis
Title
Lot Number
567333
References
Reference overview
Bigalke, H. and Shoer, L.F. 2000. In: Handbook of Experimental Pharmacology,145, Bacterial Protein Toxins (K. Aktories and I. Just, Eds.) pp. 407-443, Springer-Verlag, Berlin. Schiavo, G., et al. 1993. J. Biol. Chem.268, 23784.
Data Sheet
Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.
Revision
28-February-2012 JSW
Description
A synthetic peptide, fluorescence resonance energy transfer (FRET) substrate containing the N-terminally-linked fluorophore o-aminobenzoic acid (Abz) and the acceptor chromophore 2,4-dinitrophenol (DNP) linked to the C-terminus. The substrate is based on the native botulinum toxin type A cleavage site found in the synaptosome-associated protein SNAP-25. Cleavage of the substrate by botulinum toxin releases the fluorophore and full fluorescence is restored.
Form
Lyophilized
Formulation
Lyophilized from water.
Recommended reaction conditions
The FRET assays are performed using HEPES buffers prepared by titrating the free acid form of HEPES with the potassium salt form of HEPES. For assays with BoNT/A, the SNAPtide® stock solution is diluted using 20 mM HEPES, pH 8.0, prior to use. For assays with BoNT/A Light Chain, the stock solution should be diluted in the hydrolysis buffer, described in the section below. When using a 96-well plate and a final volume of 250 µl/well, a 250 µM stock solution is convenient to use. The final concentration of SNAPtide® to be used is typically between 5 µM and 10 µM/well, depending on the instrumentation and experiment. Since DMSO inhibits cleavage, final concentrations must be less than 2% of the total volume. For SNAPtide® (o-Abz/Dnp any concentration of ZnCl2 in the BoNT/A Light Chain hydrolysis buffer inhibits cleavage.
These FRET assays are run at 37°C. Excitation wavelength is 320 nm and emission is 420 nm. There is a linear dependence of fluorescence intensity on concentration of totally cleaved substrate up to 30 ~M SNAPtide®(o-Abz/Dnp). When measuring kinetic parameters such as the Km and Vmax for this FRET substrate, the data must be corrected for a phenomenon known as the "inner filter effect". This effect, as well as a method to determine an appropriate correction factor, is explained in the paper by Liu, et al. 1999. Analytical Biochemistry267, 331. The correction method uses an unquenched fluorophore for comparison.
Purity
≥90% by HPLC
Solubility
DMSO (5 mM). Prepare a 5 mM stock solution of this peptide in DMSO as follows: Add 40 µl of DMSO to a vial containing 200 nmol of peptide.
Storage
+2°C to +8°C
Do Not Freeze
Ok to freeze
Special Instructions
Following reconstitution, aliquot, cover with foil to protect from light, and freeze (-20°C). Stock solutions are stable for up to 3 months at -20°C.
Toxicity
Standard Handling
References
Bigalke, H. and Shoer, L.F. 2000. In: Handbook of Experimental Pharmacology,145, Bacterial Protein Toxins (K. Aktories and I. Just, Eds.) pp. 407-443, Springer-Verlag, Berlin. Schiavo, G., et al. 1993. J. Biol. Chem.268, 23784.
