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| Download: | | MSDS PDF | |
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Description:
SNAP-Cell® 430 is a blue fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells, on cell surfaces, or in vitro. This cell-permeable substrate (BG-430) is based on diethylaminocoumarin and is suitable for appropriate blue lasers and filter sets. It has an excitation maximum at 421 nm and emission maxima at 444 and 484 nm. This package includes 50 nmol of SNAP-Cell 430 substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.
The SNAP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivates of benzyl guanine. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.
There are two steps to using this system: sub-cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate is described below.
Materials required but not Supplied:
Cells expressing SNAP-tag fusion proteins
Tissue culture materials and media
Transfection reagents
Fluorescence microscope with suitable filter set
DMSO
Live CHO-K1 expressing nuclear histone H2B-SNAP (H2B-SNAP) were labeled with SNAP-Cell™ 430 (aqua).
Figure 1: Structure of BG-430 (MW 513.5)
Figure 2: Excitation (dotted line) and emission spectra of BG-430 coupled to SNAP-tag in buffer at pH 7.5
Reaction & Storage Conditions
Storage Temperature:
-20°C
Notes
Usage notes:
- Storage: SNAP-Cell 430 should be stored at -20°C (long term) or at 4°C in the dark (short term, less than 4 weeks). Protect the substrate from light and moisture. With proper storage at –20°C the substrate should be stable for at least two years dry or 3 months dissolved in DMSO.
Protocols
Protocols for SNAP-Cell® 430
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Purity and Characterization::
Purity of SNAP-Cell 430 was determined to be 88% by HPLC analysis. Molecular weight [M+H]+ was determined by MS to be 514.2 (514.2 expected).
In vitro Protein Labeling::
Reaction of SNAP-Cell 430 (10 µM) with purified SNAP-tag protein (5 µM) in vitro, followed by mass spec analysis, indicated an efficiency of labeling of 95%.
Cellular Protein Labeling::
Cells transfected with SNAP-tag vectors expressing H2B (intracellular) were labeled with 5 µM SNAP-Cell 430 for 30 minutes, and visualized by confocal microscopy. Only intracellular targets were efficiently labeled.
Legal
Licenses/Patents/Disclaimers:
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT PURPOSES ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road Ipswich, MA 01938. For detailed information, see www.neb.com/cia/legal/.