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  • Expression of ACP-ADRβ2 Fusions (N9321)

    Protocol

    1. Transient Expression
      Expression of ACP-ADRβ2 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. pACP-ADRβ2 has performed well in transient transfection of CHO-K1, COS-7, U-2 0S and NIH 3T3 cells. In most cases, ACP-ADRβ2 can be observed within 24 hours post-transfection. ACP-ADRβ2 fusion protein gives a cell surface localized signal when labeled with CoA substrates. Note that the intensity of the fluorescence may vary, depending on the cell line and labeling substrate used. This plasmid is suitable for stable transfection.

      We recommend using TransPass D2 (NEB #M2554) in combination with TransPass V (NEB #M2561) or Roche's FuGENE® 6 Transfection Reagent for both transient and stable transfections.

      Stable Expression
      pACP-ADRβ2 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 ACP-tag expression. In addition, clonal cell lines can be isolated and characterized if desired.

      Troubleshooting

      Expression
      In general, we have not experienced problems expressing ACP-ADRβ2 from the pACP-ADRβ2 plasmid. Labeling of transfected cells with a fluorescent CoA substrate should show strong surface fluorescence. Note that the intensity of this fluorescence may vary depending on cell-line and substrate used. In most instances, difficulties in expression can be resolved by altering the transfection protocol.