Solid Phase Synthesis of peptides includes Fmoc synthesis, peptide cleavage and precipitation. The synthesized peptides are lyophilized for storage.

Peptide Synthesis

  1. The peptide synthesizer is switched on.

  2. The amount of waste and solvent is checked.

  3. To ensure nitrogen is circulating and there is no leak in the system,the flowmeter is checked. Flowmeter is placed in the top right corner of the instrument.

  4. Using the program interface of the instrument, the reagent bottles are pressurized and primed.

  5. The amino acid vials are prepared with appropriate amount of aminoacid along with commensurate activating agent (HCTU/HBTU) based on the chosen scale and excess. Excess sheet is used to determine the values of amino acids and activators to be used.

  6. The aminoacid vials are loaded backward according to the desired sequence.

  7. Appropriate amount of resin is measured into one of the reaction vessels.

  8. Reaction vessels are secured in its position.

  9. A program is made (or existing program is used) to synthesize the peptide.

  10. At the end of synthesis, the empty amino acid vials are unloaded. Reaction vessel is unmounted and the contents collected.

  11. The reaction vessel is washed and cleaned before replacing it the reaction-vessel-top. Bottles are depressurized.

NOTE: For detailed steps for using peptide synthesizer, refer to the Protein Technologies Tribute peptide synthesizer manual.

Cleavage from Resin and Peptide Precipitation

  1. A cleavage cocktail is prepared by adding 95:2.5:2.5 of TFA:water:TIPS.

  2. The resin is slurried in the cleavage cocktail and the cleaving reaction is allowed to complete (generally it takes about 2 hours).

  3. The resin is separated using a syringe filter.

  4. Cold ether is added to the solution to precipitate the peptide. It can either be left overnight or centrifuged to separate the peptide completely.

  5. The supernatant is removed by decantation and disposed as waste.

Lyophilization of Peptide

  1. The Refrigerated Vapor Trap is switched on.

  2. To ensure that the fan is working, it is ensured that air is sucked in through the vent on the right side. If not, instrument is switched off immediately.

  3. The Refriferated Vapor Trap should kept on for at least 90 minutes to allow it to reach the operating temperature.

  4. Sample tubes are placed in rotor ensuring that the load is balanced.

  5. The vacuum, runtime and temperature is set.

  6. Vacuum pump and vacuum pump oil filter is switched on.

  7. The temperature and run time is set according to the peptide sequence and quantity.