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  • Expression of CLIPf-H2B (N9218)

    Protocol

    1. Transient Expression
      Expression of the fusion protein cloned in pCLIPf-H2B 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-H2B has 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-H2B 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 H2B-CLIPf from the pCLIPf-H2B plasmid. Labeling of transfected cells with a fluorescent CLIP-Cell substrate should show strong nuclear fluorescence. In most instances, difficulties in expression can be resolved by altering the transfection protocol.