Fab Purification by FPLC

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:

1. Use Sephacryl S100 column
   
2. Remove top cap, flood top surface with buffer (PBS or other)
   
3. Place column in clamps
   
4. Take top port tube, and place outlet into a waste container.
   
5. Take inlet tube 'A', dip in dH2O, and then immerse in a minimum of 300ml buffer
   
6. Run pump wash program:
   
   • Turn bottom knob to “pump wash”, press OK on dial
     
   • ~20ml of fluid will run through tubing, takes ~2min
     
7. While wash cycle is running, remove bottom plug from column and attach outlet tube
   
8. 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
     
9. 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
     
10. Turn off flow
    
    • Turn bottom knob to “End”, press OK on dial
      

Column Wash:

1. Start Computer:
   • UID: fplc_user
     
   • PW: 4020
     
2. Start “Unicorn” program on computer
   
   • UID: Moshe
     
   • PW: 123456
     
3. Select “System Control” tab on taskbar
   
4. Under 'File' menu select “Run”
   
5. Select the “Wash S100” program
   
6. Click “Next” until window with “start” appears, click “Start”
7. 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.
8. Allow wash to proceed. Wash requires 2 column volumes (240ml total), and runs at 0.5ml/min (8 hrs total run time).
9. Column is now ready for sample

Sample Addition and Separation:

1. Filter sterilize digest, using a small-volume, non-protein binding filter.
   • Alternatively, spin in a bench-top minifuge at max speed, 4oC for 20min.
2. Fill columns A-G of the collection tray with capless 1.5-2ml eppindorf tubes.
3. Place collection tray on pedestal; slot lines up with pin.
4. Stop flow, if flow did not terminate on its own.
5. Top up buffer, a minimum of 180ml is needed
6. Flush needle (located on top of control boxes) with a min of 3ml buffer
7. Inject ~3ml buffer into sample port on pump.
8. Draw sample into needle, using a clean 1ml syringe
9. Inject sample into sample port, leave needle in port as withdrawing it may create a vacuum
10. 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)
      
11. Click “next” on the box until you reach the screen with the “Start” button
12. Type in a file name for the trace file which is created
13. Click start. Run will start. Monitor fraction collector to ensure that fractions are landing in the collection tubes.
14. Run will take ~10hrs.
15. After run, use UV trace and/or western blotting to identify fractions containing the sample.
16. Western blot under non-reducing conditions to ID F(ab')2 containing fractions. Pool & concentrate for final use.

Running Another Sample:

1. Re-load sample collection tray
2. Inject 3ml of buffer into sample port, as done previously
3. Inject sample into sample port, as done previously
4. Run sample using “Ron S100” protocol
   

Preparing Column for Storage:

1. Remove input tube from top of column.
2. Immerse inlet tube 'A' in at least 300ml of 20% ethanol in ddH2O.
3. Clear input tube an pump, using the pump clear protocol described in steps 4-10 of “Column Preparation”
4. Clean the column, as per the “Column Wash” portion of this protocol, using 20% ethanol in place of buffer.
5. Once wash cycle is complete, remove input/output tubes, cap column, and store column behind the column stand.