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  • Expression of ACP-tag Fusions (N9322)

    Protocol

    1. Transient Expression
      Expression of the fusion protein cloned in pACP-tag(m)-2 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.

      ACP-tag fusion proteins can be observed 24 hours post-transfection. We recommend using pACP-ADRβ2 as an expression control plasmid. ACP-ADRβ2 fusion protein gives a cell surface localized signal when labeled with CoA substrates.

      pACP-ADRβ2 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 the cell line and labeling substrate used.

      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-tag(m)-2 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 ACP-tag expression. In addition, monoclonal cell lines can be isolated and characterized, if desired. 

      Troubleshooting 

      Expression 
      In general, we have not experienced problems expressing ACP-tag protein fusions. However, if the fusion gene does not appear to be expressed, try expressing the ACP-ADRβ2 protein fusion as a positive control using cells transiently transfected with the pACP-ADRβ2 Control Plasmid (NEB #N9321 ). Labeling of such cells with a fluorescent CoA substrate should show strong membrane fluorescence. Note that the intensity of this fluorescence may vary depending on cell-line and substrate used.

      If the pACP-ADRβ2 construct is 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 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.