SNAP-Cell® Fluorescein is a green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells or in 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 small protein based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylpurines and benzylchloropyrimidines, In the labeling reaction, the substituted benzyl group of the substrate becomes 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.
Figure 1:Live CHO-K1 cells transiently transfected with pSNAP-H2B
Cells were labeled with SNAP-Cell Fluorescein (green) for 15 minutes.Figure 2: Excitation (dotted line) and emission spectra of SNAP-Cell Fluorescein (fluorescent product of SNAP-Cell Fluorescein in cells) coupled to SNAP-tag in buffer at pH 7.5)
Figure 3: Structure of SNAP-Cell Fluorescein (MW 712.7g/mol)
Notes:
Storage: SNAP-CellFluorescein 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 andmoisture. With proper storage at -20°C the substrate should be stablefor at least 3 years dry or 3 months dissolved in DMSO.
