Flu-P1: Ligating the peptide to the C-terminus of a thioester-tagged protein
- Combine 10 µl of the resuspended Flu-P1 peptide with 40 µl of protein purified using the IMPACT-TWIN or IMPACT-CN Systems. It is important to use a C-terminal intein fusion and MESNA to induce intein cleavage during protein purification (see the FAQs pertaining to IPL in the IMPACT Kits or on www.neb.com website). We recommend incubating the Flu-P1 peptide and thioester-tagged protein overnight at room temperature. If the target protein is unstable at room temperature, use an overnight incubation at 4°C. If the unreacted Flu-P1 peptide interferes with subsequent experimentation it can be separated from the protein by FPLC using a column appropriate for the target protein.