SNAP-Cell 647-SiR is a far-red fluorescent substratethat can be used to label SNAP-tag® fusion proteinsinside living cells, on cell surfaces, or in vitro. Thiscell-permeable substrate (also termed SiR-SNAP)(1)is based on 6-carboxy-tetramethylsiliconrhodamineand is suitable for standard Cy5 filter sets. It hasan excitation maximum at 645 nm and an emissionmaximum at 661 nm. This package contains 30 nmolof SNAP-Cell 647-SiR substrate, sufficient to make10 ml of a 3 μM SNAP-tag fusion protein labelingsolution.
The SNAP-tag protein labeling system enables thespecific, covalent attachment of virtually any moleculeto a protein of interest. The SNAP-tag is based onhuman O6-alkylguanine-DNA-alkyltransferase (hAGT).SNAP-tag substrates are fluorophores, biotin or beadsconjugated to guanine or chloropyrimidine leavinggroups via a benzyl linker. In the labeling reaction, thesubstituted benzyl group of the substrate is covalentlyattached to the SNAP-tag.
There are two steps to using this system: subcloningand expression of the protein of interest as aSNAP-tag fusion, and labeling of the fusion with theSNAP-tag substrate of choice. Expression of SNAPtagfusion proteins is described in the documentationsupplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate isdescribed below.
Figure 1. Live NIH/3T3 cells expressing SNAP-tag fused to mitochondrial oxidase 8A (Cox8A-SNAPf)
Cellswere labeled with SNAP-Cell 647-SiR (red) for 30minutes at 37°C, 5% CO2.Figure 2: Excitation (dotted line) and emission spectra of SNAP-Cell 647-SiR after coupling to SNAP-tag in buffer at pH 7.5 Figure 3: Structure of SNAP-Cell 647-SiR (MW 724.9 g/mol)
Notes:
Storage:SNAP-Cell 647-SiR should be stored at -20°C(long term) or at 4°C in the dark (short term, lessthan 4 weeks). Protect the substrate from lightand moisture. With proper storage at -20°C thesubstrate should be stable for at least three yearsdry or 3 months dissolved in DMSO.
