Recombinant GFP/SNAP25B/VAMP-2 Protein, CF

Catalog #: 7375-SV Datasheet / COA / SDS

Substrate for Botulinum and Tetanus Neurotoxin Proteases

Catalog #	Availability	Size / Price	Qty
7375-SV-050

R&D Systems Recombinant Proteins and Enzymes

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All GFP/SNAP25B/VAMP-2 Proteins and Enzymes

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

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

Product Specifications

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.

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.

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
Analysis

Val (of MVKSADI)

Predicted Molecular Mass

52 kDa

SDS-PAGE

54-58 kDa, reducing conditions

Product Datasheets

7375-SV

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

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.

Assay Procedure

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

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.

Per Lane:

rBoNT/D-LC: 4 ng
Substrate: 1 µg

Reconstitution Calculator

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