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  • Expression of CLIPf-NK1R (N9216)

    Protocol

    1. Transient Expression
      Expression of the fusion protein cloned in pCLIPf-NK1R 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. pCLIPf-NK1R 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
      pCLIPf-NK1R can be transfected as described above for transient transfection or by other 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 clonal cell lines can be isolated and characterized if desired.

      Troubleshooting

      Expression
      In general, we have not experienced problems expressing CLIPf-NK1R from the pCLIPf-NK1R plasmid. Labeling of transfected cells with a fluorescent CLIP-Cell or CLIP-Surface substrate should show strong cell surface fluorescence. In most instances, difficulties in expression can be resolved by altering the transfection protocol.