Recombinant GFP/SNAP25B/VAMP-2 Protein, CF

R&D Systems | Catalog # 7375-SV

Substrate for Botulinum and Tetanus Neurotoxin Proteases
R&D Systems
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Key Product Details

  • R&D Systems E. coli-derived Recombinant GFP/SNAP25B/VAMP-2 Protein (7375-SV)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

ACS44347 (GFP); P60880 (SNAP25B); P63027 (VAMP-2)

Applications

Enzyme Activity
View all GFP/SNAP25B/VAMP-2 Proteins and Enzymes »

Product Specifications

Source

E. coli-derived GFP/SNAP25B/VAMP-2 protein
MVKSADI 10-His tag Linker 1 GFP
(Ser2-Lys238)
Accession # ACS44347		 Linker 2 Human SNAP25B
(Asn93-Gly206)
Accession # P60880		 Human VAMP-2
(Ser2-Lys94)
Accession # P63027		 Cys
N-terminus C-terminus

Purity

>85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Val (of MVKSADI)  

Predicted Molecular Mass

52 kDa

SDS-PAGE

54-58 kDa, reducing conditions

Activity

Measured by cleavage by botulinum and tetanus toxin light chains.
>50% of 1 μg can be cleaved by 4 ng of toxin light chain, as measured under the described conditions.

Formulation, Preparation, and Storage

7375-SV
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Tween®.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: GFP/SNAP25B/VAMP-2

Botulinum and tetanus neurotoxins (BoNTs and TeNT) are zinc metalloproteases that hydrolyze and inactivate proteins necessary for neurotransmission. The protease domain is located in the light chain of the neurotoxin. Among the known substrates of BoNTs and TeNT are synaptosomal protein-25 (SNAP25) and vesicle-associated membrane protein (VAMP). This substrate incorporates a green fluorescent protein (GFPuv) and portions of human SNAP25B and VAMP‑2 that contain the cleavage sites of all the known BoNTs and TeNT. The light chains of BoNTs -A, -C, and -E cleave the SNAP25B sequence, while BoNTs -B, -D, -F, -G, and TeNT cleave within the VAMP‑2 sequence.  The substrate can be used in a SDS‑PAGE gel-shift assay to detect cleavage by the neurotoxin proteases. Alternatively, the substrate can be coupled to maleimide-activated microwell plates through the C-terminal cysteine residue to generate a high throughput assay format. A similar substrate and assay format has been used to screen for inhibitors of these neurotoxin proteases (1).

References

  1. Hines, H.B. et al. (2008) Applied and Environ. Microbiol. 74:653.

Long Name

Green Fluorescent Protein:SNAP25B/VAMP-2

Alternate Names

GFP, Green Fluorescent Protein

UniProt

ACS44347 (GFP); P60880 (SNAP25B); P63027 (VAMP-2)

Additional GFP/SNAP25B/VAMP-2 Products

Product Documents for Recombinant GFP/SNAP25B/VAMP-2 Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant GFP/SNAP25B/VAMP-2 Protein, CF

For research use only

Citations for Recombinant GFP/SNAP25B/VAMP-2 Protein, CF

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Protocols

View specific protocols for Recombinant GFP/SNAP25B/VAMP-2 Protein, CF (7375-SV):

Materials
  • Assay Buffer: 50 mM HEPES, pH 6.5
  • Recombinant C. botulinum BoNT-D Light Chain (rBoNT/D-LC) (Catalog # 6037-ZN)
  • Substrate: Recombinant GFP/SNAP25B/VAMP-2 (R&D Systems, Catalog # 7375-SV)
  • SDS-PAGE and silver stain reagents or Western blot with appropriate antibodies
  1. Dilute Substrate to 100 µg/mL in Assay Buffer.
  2. Dilute rBoNT/D-LC to 0.4 µg/mL in Assay Buffer.
  3. Combine equal volumes of diluted Substrate with diluted rBoNT/D-LC. Prepare two controls by combining equal volumes of diluted Substrate with Assay Buffer.
  4. Incubate reaction vials at room temperature for 1 hour. Incubate one control at room temperature and the other at -20 °C for 1 hour.
  5. After incubation, combine reaction mixtures and controls with reducing SDS-PAGE sample buffer at a 2:1 (reaction mixture:sample buffer) ratio (v/v) to stop reactions.
  6. Analyze the cleavage products by SDS-PAGE (Load 30 µL of the mixture from step 5 per lane (1 µg Substrate per lane) followed by silver staining and/or Western blot.

Per Lane:

  • rBoNT/D-LC: 4 ng
  • Substrate: 1 µg

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