FPLC Checklist

Contents

1. Before you begin
2. Beginning a purification run
3. Finishing a purification run

1. Before you begin

Always filter all FPLC buffers using a 0.45 or smaller µm filter, degas, and chill to 4°C to avoid bubbles and clogging

1. Treat your lysate with DNAse and use the Ultracentrifuge to clarify.
2. Perform system and pump washes with water to remove ethanol. The salt in purification buffers will crash out when exposed to ethanol and can destroy columns.
3. Purge the pumps to remove air from the pump heads.

2. Beginning a purification run

1. Set alarms for pre column and delta column pressures according to the column manual.
2. Perform “Pump Wash A & Pump Wash B” with water.
3. Wash column with ~10 column volumes (CV) of water. Set pause timer.
4. Wash column with ~10 CV of buffer B to flush out residual protein from a previous prep. Set pause timer.
5. Wash column with ~10CV of buffer A. Set pause timer.

3. Finishing a purification run

If running manually, set appropriate time/volume pause timers to avoid running the system dry.

1. Wash column with 100% Buffer B to ensure protein is completely eluted.
2. Wash column with H2O.
3. Store column in water (for up to 48 hours) or 20% EtOH (>48 hours between use) and label the column with the storage solution, the date, and your initials.
4. Clean the syringe port with HCl, NaOH, and water.
5. Perform a system wash with water and then with 20% EtOH.
6. Empty the fraction collector.
7. Wipe the dispenser head and sensor diode of the fraction collector with an ethanol-soaked Kimwipe.