Cleavage procedure To cleave peptide from 50 mg of resin (to cleave a small amount for MALDI reduce all volumes

by 3): 1) Place resin in a clean sample vial and syringe in 2.85 ml of TFA, 0.15 ml of water, then 0.15 ml of triisopropylsilane (TIPS). Place a lid on the vial, swirl, and leave to stand for at least two hours with occasional swirling. 2) Following cleavage of resin and protecting groups, filter out resin beads. This can be achieved by use of a filter formed from a small piece of cotton wool or tissue paper pushed into a small pipette. A second pipette is used to transfer the TFA mixture into the improvised filter. When the solution has run through into a clean sample vial, no resin should remain. 3) The TFA and water is removed by use of rotary evaporation at 47 C and ~10 mbar pressure. 4) Diethylether is added (6 ml) to assist with total removal of TFA by rotary evaporation under the conditions used previously. 5) When only a wet solid remains, 6 ml more ether is added to precipitate out the peptide. A pipette is used to scrape any peptide off the glass in order to suspend the peptide in ether. Upon leaving the flask to enable the mixture to separate, the ether can be removed from peptide (a white solid settled at the bottom of the flask) by use of careful pipetting (it can be easier to take liquid from the sides of the flask rather then the centre as this minimises disturbance to the peptide). 6) Finally, 3 ml more ether is added, and removed by rotary evaporation to ensure complete removal of TFA and ether. The peptide should be isolated as a white solid, sealed and stored under refrigeration.