Expression of ACP Fusions (N9135)
- Transient Expression
Expression of the fusion protein cloned in pACP 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.
In general we have not experienced problems ex-pressing ACP-tag protein fusions. However if your fusion gene does not appear to be expressed, try expressing the pACP-GPI control plasmid (NEB # N9319S). Labeling of such cells with a fluorescent ACP-tag substrate should show membrane fluorescence. Note that the intensity of this fluorescence may vary depending on cell-line and substrate used.
If the ACP-GPI construct is expressed but your fusion protein is not, then there are a variety of possible causes. It is possible that this fusion protein may be toxic for your cell line. It is difficult to trou-bleshoot 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.