SNAP-Cell Oregon Green is a photostable green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells. This cell-permeable substrate (BG-Oregon Green) is based on the Invitrogen dye, Oregon Green and is suitable for standard fluorescein filter sets. It has an excitation maximum at 490 nm and an emission maximum at 514 nm. Conjugates of Oregon Green are more photostable than those of fluorescein, and their fluorescence properties are essentially pH insensitive in the physiological pH range. This package contains 50 nmol of SNAP-Cell Oregon Green 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 based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylguanines and benzylchloropyrimidines. In the labeling reaction, the dye-substituted benzyl group of the substrate becomes 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.
Figure 1: Live U2OS cells transiently transfected with pSNAP-Cox8A (mitochondrial expression).
Cells were labeled with SNAP-Cell Oregon Green (green) for 15 minutes and counterstained with Hoechst 33342 (blue).Figure 2: Excitation (dotted line) and emission spectra after coupling of SNAP-Cell Oregon Green to SNAP-tag in buffer at pH 7.5.
Notes:
Storage:SNAP-Cell Oregon Green 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 three years dry or 3 months dissolved in DMSO.SNAP-Cell Oregon Green is not suitable for labeling of cell surface SNAP-tag fusion proteins. The Oregon Green dye is based on a fluorinated fluorescein modified with pivaloyl groups which is uncharged and essentially non-fluorescent. It becomes fluorescent once inside the cell where it is hydrolyzed by non-specific esterases.
