Fab Purification by FPLC
Jump to navigation
Jump to search
Notes
- All buffers must be filtered or otherwise rendered particle-free.
- pH 2-10 can be used
- Sterile PBS can be used as a buffer.
- A maximum of 2ml can be run through the column at any one time
- Sample must be filtered or otherwise rendered particle-free
- Sample should be as concentrated as possible, 1mg/ml or better
- Roomkey: 3524
Protocol
Column Preparation:
- Use Sephacryl S100 column
- Remove top cap, flood top surface with buffer (PBS or other)
- Place column in clamps
- Take top port tube, and place outlet into a waste container.
- Take inlet tube 'A', dip in dH2O, and then immerse in a minimum of 300ml buffer
- Run pump wash program:
- Turn bottom knob to “pump wash”, press OK on dial
- ~20ml of fluid will run through tubing, takes ~2min
- Turn bottom knob to “pump wash”, press OK on dial
- While wash cycle is running, remove bottom plug from column and attach outlet tube
- When wash is done, run pump at slow speed (~0.5ml/min)
- Turn bottom knob to “Set Flow Rate”, press OK on dial
- Turn bottom knob to 0.5ml/min, press OK on dial
- Turn bottom knob to “Set Flow Rate”, press OK on dial
- Once drops emerge from inlet tube, attach to upper port of column
- A drop from the inlet tube should be touched to the layer of buffer on the column to avoid air bubbles
- A drop from the inlet tube should be touched to the layer of buffer on the column to avoid air bubbles
- Turn off flow
- Turn bottom knob to “End”, press OK on dial
- Turn bottom knob to “End”, press OK on dial
Column Wash:
- Start Computer:
- UID: fplc_user
- PW: 4020
- UID: fplc_user
- Start “Unicorn” program on computer
- UID: Moshe
- PW: 123456
- UID: Moshe
- Select “System Control” tab on taskbar
- Under 'File' menu select “Run”
- Select the “Wash S100” program
- Click “Next” until window with “start” appears, click “Start”
- Check column depositor to ensure buffer is running into the waste container.
- If no flow occurs after a few minutes there is likely an air bubble in the system. This should also cause an over-pressure alarm and automatic shutdown of the wash column program.
- Allow wash to proceed. Wash requires 2 column volumes (240ml total), and runs at 0.5ml/min (8 hrs total run time).
- Column is now ready for sample
Sample Addition and Separation:
- Filter sterilize digest, using a small-volume, non-protein binding filter.
- Alternatively, spin in a bench-top minifuge at max speed, 4oC for 20min.
- Fill columns A-G of the collection tray with capless 1.5-2ml eppindorf tubes.
- Place collection tray on pedestal; slot lines up with pin.
- Stop flow, if flow did not terminate on its own.
- Top up buffer, a minimum of 180ml is needed
- Flush needle (located on top of control boxes) with a min of 3ml buffer
- Inject ~3ml buffer into sample port on pump.
- Draw sample into needle, using a clean 1ml syringe
- Inject sample into sample port, leave needle in port as withdrawing it may create a vacuum
- Start unicorn, run the “Ron S100” program
- Flow rate should be 0.25ml/min
- Load volume should be 1.2ml
- Collection Volume should be 1.2ml/tube
- Elution volume should be 1.5 column volumes (180ml)
- Flow rate should be 0.25ml/min
- Click “next” on the box until you reach the screen with the “Start” button
- Type in a file name for the trace file which is created
- Click start. Run will start. Monitor fraction collector to ensure that fractions are landing in the collection tubes.
- Run will take ~10hrs.
- After run, use UV trace and/or western blotting to identify fractions containing the sample.
- Western blot under non-reducing conditions to ID F(ab')2 containing fractions. Pool & concentrate for final use.
Running Another Sample:
- Re-load sample collection tray
- Inject 3ml of buffer into sample port, as done previously
- Inject sample into sample port, as done previously
- Run sample using “Ron S100” protocol
Preparing Column for Storage:
- Remove input tube from top of column.
- Immerse inlet tube 'A' in at least 300ml of 20% ethanol in ddH2O.
- Clear input tube an pump, using the pump clear protocol described in steps 4-10 of “Column Preparation”
- Clean the column, as per the “Column Wash” portion of this protocol, using 20% ethanol in place of buffer.
- Once wash cycle is complete, remove input/output tubes, cap column, and store column behind the column stand.