Benzylguanine (BG) derivatives of Spirochrome’s SiR and SPY™ probes are now available for self-labeling SNAP-tag™* substrates.
Spirochrome’s BG derivatives emit between the orange and far-red UV-ViS spectrum (Figure 1) while retaining the appealing traits of their non-BG counterparts. Spirochrome’s BG derivatives are fluorogenic, highly cell permeable, and are well suited for conventional or super resolution microscopy. One major advantage of SNAP-tag labeled proteins is the strong and stable covalent thioether bond that is not easily disrupted. These irreversible bonds create a permanent fusion between the protein of interest and the substrate even when in protein homogenates or lysates. Thus, SNAP-tagged proteins can be even loaded, detected, and quantitated with in-gel fluorescence scanning. Further, SNAP-tag fusions are versatile and can be appended to the N- or C- protein terminus avoiding disruptions in biological function.
In addition to labeling the cellular surface, SNAP-tag labeling can label internal cellular structures or proteins even if located within the nucleus and ER Golgi; however, cell permeable substrates are required. Fortunately, cell permeable SPY™ and SiR probes work synergistically to label subcellular structures even internal proteins within the mitochondrial cristae1. Being compatible with STED and SIM super resolution imaging even further augments the utility of SPY™ and SiR probes, especially when analyzing spatio-temporal protein dynamics. Once cells are stably expressing fusion SNAP-tags they can be labeled by Spirochrome BG derivatives (Figure 2).
The use of BG substrates requires a suitable SNAP-tag fusion protein. Kits to produce SNAP-tag fusion proteins can be purchased from other suppliers. Once cells or tissues are stably expressing SNAP-tagged proteins, Spirochrome’s BG derivatives can be used to label expressed SNAP-tags. The general workflow and protocol are outlined below.
*SNAP-tag™ is a registered trademark of New England Biolabs, Inc.
Labeling organellar proteins with BG derivatives require that the protein of interest have an attached SNAP-tag for covalent attachment, and that the fluorophore of your derivative be cell permeable. Fortunately, SPY and SiR BG derivatives are highly cell permeable and have been used to label mitochondrial proteins1.
SNAP-tag™ is a registered trademark of New England Biolabs, Inc. Requests for guidance with the creation of fusion genes and subsequent proteins should be directed to NEB.
Internal testing shows that HeLa cells stably express label-ready SNAP-tags after 48 hours when using Lipofectamine 3000.