SNAP-Cell® Fluorescein is a green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells orin vitro. This cell-permeable substrate (BG-Fluorescein) is based on diacetylfluorescein and is suitable for standard fluorescein filter sets. Diacetylfuorescein is essentially non-fluorescent, but it becomes fluorescent inside the cell when it is hydrolyzed by non-specific esterases, yielding fluorescein. It has an excitation maximum at 500 nm and an emission maximum at 532 nm. This substrate has limited photostability. If this presents a problem, we recommend using SNAP-Cell 505, which has similar spectral characteristics but much greater photostability. This package contains 50 nmol of SNAP-Cell Fluorescein 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: 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 fusion proteins with the SNAP-tag substrate is described below.
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