SNAP-Surface® 682 is a photostable fluorescent substrate that can be used to label SNAP-Tag® fusion proteins in solution. This substrate (BG-682) is based on the Dyomics dye DY-682 and is suitable for excitation with a 680 nm diode laser. It has an excitation maximum at 693 nm and emission maxima at 708 nm. This package includes 50 nmol of BG-632 substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.
The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a protein based on human O6-alkylguanine-DNA alkyltransferase (hAGT). SNAP-tag substrates are fluorophores, biotin or beads conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. 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: subcloning 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-fusion proteins. Proteins of interest can be expressed with the SNAP-tag as either an N- or a C-terminal fusion, but note that the tag needs to be exposed to the extracellular surface of the plasma membrane for labeling with SNAP-Surface 682.
• Tissue culture materials and media
• Transfection reagents
• Fluorescence microscope with suitable filter set
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