Expression of SNAP Fusions (N9172)

Protocol

1. Transient Expression
   Expression of the fusion protein cloned in pSNAP-tag(m) can be achieved by transiently transfecting cells in culture with standard transfection protocols. The appropriate reagent and time to permit adequate expression must be empirically determined. We recommend using pSNAP-H2B or pSNAP-Cox8A as expression control plasmids. H2B-SNAP fusion protein gives a nuclear localized signal and the COX8-2-SNAP fusion protein gives a mitochondrial localized signal when labeled with SNAP-cell substrates. If the empty pSNAP-tag(m) plasmid is used as a control vector for transfection, an even distribution of the SNAP-tag between nucleus and cytoplasm should be seen. Both pSNAP-tag(m) and the localization control plasmids have performed well in stable and transient transfection of CHO-K1, COS-7, U-2 OS and NIH 3T3 cells. Note that the intensity of the fluorescence may vary depending on cell line and labeling substrate used. 
2. Stable Expression
   pSNAP-tag(m) and the localization control plasmids can be transfected by standard transfection methods. Twenty four to 48 hours after transfection begin selecting mammalian cultures in 600–1,200 µg/ml G418 (geneticin) depending on the cell line. It is recommended that you establish a kill curve for each cell line to determine optimal selection conditions. After 8–12 days of continuous selection, stable colonies will become visible. It is possible to use pools of stable cell populations for initial cell labeling to test for the presence of SNAP-tag expression. In addition monoclonal cell lines can be isolated and characterized if desired.
3. Troubleshooting: Expression
   In general we have not experienced problems expressing SNAP-tag protein fusions. However if your fusion protein does not appear to be expressed, try expressing the H2B-SNAP or COX8-2-SNAP protein fusions as positive controls using cells transiently transfected with pSNAP-H2B or pSNAP-Cox8A. Labeling of such cells with a fluorescent SNAP-Cell substrate should show strong nuclear or mitochondrial localized fluorescence, respectively. The empty pSNAP-tag(m) plasmid can also be used as a control (cytosolic and nuclear fluorescence). Note that the intensity of this fluorescence may vary depending on cell line and substrate used. If the localization controls are expressed but your fusion protein is not, then there are a variety of possible causes. It is possible that this fusion protein may be toxic for your cell line. It is difficult to troubleshoot such instances, but the use of a different expression plasmid or cell line or tagging the opposite end of the protein may help. Signs of host cell toxicity could include slow proliferation or apoptosis. Counterstaining live cells with Hoechst 33342 or fixed cells with DAPI can be used to determine whether nuclei are healthy if toxicity is suspected.