Expression of MCP Fusions (N9317)
Protocol
- Transient expression
Expression of the fusion protein cloned in pMCP-tag(m) Vector 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.
- Troubleshooting
In general we have not experienced problems expressing MCP-tag protein fusions. However if your fusion gene does not appear to be expressed, try expressing the pMCP-GPI Control Plasmid (NEB #N9320S ). Labeling of such cells with a fluorescent CoA Substrate should show membrane fluorescence. Note that the intensity of this fluorescence may vary depending on cell line and substrate used.
If the MCP-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 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.
