Expression of CLIP Fusions (N9211)
Protocol
- Transient Expression
Expression of the fusion protein cloned in pCLIP-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 pCLIP-H2B or pCLIP-NK1R as expression control plasmids. H2B-CLIP fusion protein gives a nuclear localized signal and the CLIP-NK1R fusion protein gives a membrane localized signal when labeled with CLIP-Cell substrate. If the empty pCLIP-tag(m) plasmid is used as a control vector for transfection, an even distribution of the CLIP-tag between nucleus and cytoplasm should be seen. Both pCLIP-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. - Stable Expression
pCLIP-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 CLIP-tag expression. In addition monoclonal cell lines can be isolated and characterized if desired. - Troubleshooting
In general we have not experienced problems expressing CLIP-tag protein fusions. However if your fusion protein does not appear to be expressed, try expressing the H2B-CLIP or CLIP-NK1R protein fusions as positive controls using cells transiently transfected with pCLIP-H2B or pCLIP-NK1R. Labeling of such cells with a fluorescent CLIP-Cell™ substrate should show strong nuclear fluorescence in case of H2B-CLIP or membrane localized fluorescence in case of CLIP-NK1R when using CLIP-Cell substrates. The empty pCLIP-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.
