Sauer:FPLC protocol
How to use the FPLC
Preparation:
- Sign up for FPLC.
- Prepare H2O and 20% EtOH (unless there is already plenty)
- Both must be filtered and degassed.
- Prepare buffers
- Must be filtered and degassed.
Attach column:
4) Set flow rate to ~0.5mL/min
5) Take off top of column, to minimize air entering column, ensure the top of the column is wet via the syringe at the bottom.
6) Connect directly to flow
7) Remove syringe and attach base of column in to UV detector
Running:
8) Column is stored in 20% EtOH, so start by washing in into dH2O. if a pump is not already in water, use pumpwash to flush pump over to dH2O.
9) Pumpwash into your buffer.
10) Wash column with buffer
11) Do your run.
Cleaning:
12) Wash column. If you were doing a gradient, wash it with high salt for a few. If isocratic, you may need to wash with high salt regardless or with NaOH, according to the cleaning protocol for that column. (look it up online). This does not need to be done every time, but if the column is not performing that well… hint, hint.
13) Flush column with water
14) Store column in 20% EtOH
15) Empty (or replace) waste containers, making certain to put tubing back into them once they’re empty.
How to not be murdered:
Columns **MUST** be stored in either dH2O or EtOH. They may only be stored up to 2 days in water or up to 1 day in your buffer. If you store the column with salt, the salt will crystallize and you will destroy the column. Also, the same fate as for water will occur: If you store it for long periods in water, things will grow. You will destroy the column.
Pumps should both be left in 20% EtOH. The fridge is not clean. Things grow. We have repeatedly had contamination issues in the whole system because the water had crap living in it. Prevent this.
