Expression of CLIP-tag Fusions (N9215)
- Transient Expression
Expression of the fusion protein cloned in pCLIPf 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 pCLIPf-H2B (NEB #N9218) as an expression control plasmid. H2B-CLIPf fusion protein gives a nuclear localized signal and the COX8-2-CLIPf fusion protein gives a mitochondrial localized signal when labeled with CLIP-Cell substrates. If the empty pCLIPf plasmid is used as a control vector for transfection, an even distribution of the CLIP-tag in nucleus and cytoplasm should be seen. Both pCLIPf and the localization control plasmid 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 the cell line and labeling substrate used.
pCLIPf 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 a kill curve be established 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.
In general, we have not experienced problems expressing CLIP-tag protein fusions. However, if the fusion protein does not appear to be expressed, try expressing the H2B-CLIPf or protein fusion as a positive control using cells transiently transfected with pCLIPf-H2B. Labeling of such cells with a fluorescent CLIP-Cell substrate should show strong nuclear localized fluorescence. The empty pCLIPf 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 the fusion protein is not, then there are a variety of possible causes. It is possible that this fusion protein may be toxic for the 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 (N or C) 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.