Peptide Synthesis

Goal

• Goal: To successfully synthesize high quality peptides

Pre-Protocol Questions

1. Do you know how to properly load and set up cartridges?

2. Will Karin be available on day 2 to assist in the HBTU and synthesis?

3. Do you know where the lyophilizer is and how to use it?

Protocol

Day 1: Weighing Out Peptides 1. Take A.A from the fridge to let them warm at room temperature (This prevents water from condensing into containers which may disrupt the reaction).

2. While warming, set up clean cartridges in a metal rack. To reduce deletion sequences and improve peptide quality, make sure to double couple each peptide.

3. Weigh out the amount of each amino acid (A.A) required for a 0.5 mmol reaction. Pour into labeled cartridge. DO NOT create a cartridge for the A.A on the C-terminal end of the peptide.

4. Parafilm cartridges and place back into the fridge.

• Time permitting, skip step 4 and move directly to the Day 2 Protocol.

Day 2: HBTU and Synthesis

1. Remove metal racks from the fridge and allow them to warm at room temperature.

2. Weigh out the amount of HBTU required for 0.5 mmol reaction. Place into each cartridge.

3. Cap each cartridge with a metal cap and black rubber stopper using the clamp tool.

4. Add 0.1 mmol resin to the reaction vessel. Resin varies between peptide samples. Different resins are used because they are attached to the A.A on the C-terminal end of the peptide.

5. Run peptide synthesis on the Gryos 2-Channel Peptide Synthesizer. To operate the machine:

    a. Select Load Synthesis on the peptide synthesizer to begin loading Reaction Vessel 1. 
    b. Choose first program: 0.1mmol_DC_1_swell_20min. (Remember, all peptides are loaded from the C-terminal to the N-terminal end. Thus, the first sample will be the cartridge containing the resin linked to most the C-terminal A.A. Additionally, the swell program is ONLY used on the resin sample) 
    c. Load A.A and select forward.
    d. Repeat with cycle number 2: 0.1 mol_DC_20min.
    e. Continue until all A.A are loaded
    f. If running two peptide syntheses, use manual operations to move to Reaction Vessel 2. 
    g. Repeat steps 5b - 5e. 
    h. Run reaction

NOTE: MAKE SURE KARIN IS IN THE ROOM TO ASSIST DURING THIS STEP. Remember, this is her equipment and she is eager to help operate the machine. She will load all of the solvents prior to the synthesis reaction and will manage the loading process.

6. After synthesis is complete, remove reaction vessels from the synthesizer and place in the lyophilizer (Removes excess water in the sample by sublimation).

Day 3: Cleaning Amino Acid Cartridges

NOTE: Complete all work under the hood.

1. Obtain used amino acid cartridges, empty metal rack, pliers, paper towels, and a squirt bottle full of methanol.

2. Using the pliers, remove the metal cap and rubber stopper off of the plastic cartridge. Dispose of cap and stopper in the waste basket.

3. Using the bottle, squirt methanol into the cartridge to remove excess residue. Dispose of liquid waste in a 1 liter beaker.

4. Repeat methanol wash 2-3 times or until clean. Additionally, spray a Kimwipe with methanol and use it to remove any marker labeling present on the outside of the cartridge.

NOTE: If unable to clean the inside of the cartridge, dispose of it in the waste basket.

5. To dry, place the clean cartridge top down in a metal rack (with paper towels lining the bottom).

6. Repeat steps 2-5 until all cartridges are clean.