Recombinant GFP/SNAP25B/VAMP-2 Protein, CF
Recombinant GFP/SNAP25B/VAMP-2 Protein, CF Summary
|MVKSADI||10-His tag||Linker 1||GFP
Accession # ACS44347
|Linker 2||Human SNAP25B
Accession # P60880
Accession # P63027
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|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.
- 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
- Dilute Substrate to 100 µg/mL in Assay Buffer.
- Dilute rBoNT/D-LC to 0.4 µg/mL in Assay Buffer.
- Combine equal volumes of diluted Substrate with diluted rBoNT/D-LC. Prepare two controls by combining equal volumes of diluted Substrate with Assay Buffer.
- Incubate reaction vials at room temperature for 1 hour. Incubate one control at room temperature and the other at -20 °C for 1 hour.
- 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.
- 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.
- rBoNT/D-LC: 4 ng
- Substrate: 1 µg
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).
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