SNAP-Surface® 647 is a photostable fluorescent substrate that can be used to label SNAP-tag® fusion proteins on cell surfaces and in solution. This substrate (BG-647) is based on the Dyomics dye DY-647 and is suitable for excitation with a 650 nm diode laser or use with Cy7 filter sets. It has an excitation maximum at 660 nm and emission maximum at 673 nm. This package includes 50 nmol of SNAP-Surface 647 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 647.
• Tissue culture materials and media
• Transfection reagents
• Fluorescence microscope with suitable filter set
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