Recombinant GFP/SNAP25B/VAMP-2 Protein, CF

• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant GFP/SNAP25B/VAMP-2 Protein, CF Summary

• Additional Information: Substrate for Botulinum and Tetanus Neurotoxin Proteases
• Details of Functionality: 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.
• 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

• Accession #: ACS44347 (GFP); P60880 (SNAP25B); P63027 (VAMP-2)
• N-terminal Sequence: Val (of MVKSADI)
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 52 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 54-58 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Tween®.
• Purity: >85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant GFP/SNAP25B/VAMP-2 Protein, CF

• GFP
• GFP/SNAP25B/VAMP-2
• Green Fluorescent Protein

Background

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).

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

Publications for GFP/SNAP25B/VAMP-2 (7375-SV)(1)

Publication using 7375-SV

• Albarran, E;Sun, Y;Liu, Y;Raju, K;Dong, A;Li, Y;Wang, S;S�dhof, TC;Ding, JB; Postsynaptic synucleins mediate endocannabinoid signaling Nature neuroscience 2023-05-29 [PMID: 37248337] (Bioassay, N/A)

Bioinformatics

• Uniprot:
  • ACS44347 (GFP); P60880 (SNAP25B); P63027 (VAMP-2)()