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FPLC Protocols

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Harms Lab Wiki > Protein Expression & Purification > > FPLC Protocols

Start up checklist

  1. Make sure the waste is empty.
  2. Turn on the lamp, Parameters –> Lamp (ON)
  3. Move the input lines from 20% EtOH and place them into ddH2O.
  4. Fill the input lines with ddH2O.  Go to Templates->Application Templates->System Wash Method and select lines A and B.  This will fill the input lines with ddH2O.  
  5. Place the column in line.
  6. Flush the column with 5 column volumes of ddH2O using a Manual Run.  Start at 1 mL/min, 0.5 MPa pressure limit.  Ramp to 5 mL/min over ~30 second to avoid overpressure error. 
  7. Move the input lines from ddH2O to the appropriate A and B buffers.
  8. Fill the input lines with buffer.  Go to Templates->Application Templates->System Wash Method and select lines A and B.  This will fill the input lines with A and B buffers.  
  9. Flush the column with 5 column volumes of buffer A using a Manual Run. Start at 1 mL/min, 0.5 MPa pressure limit.  Ramp to 5 mL/min over ~30 second to avoid overpressure error. 

Shut down checklist

  1. Move the input lines into ddH2O.
  2. Fill the input lines with ddH2O.  Go to Templates->Application Templates->System Wash Method and select lines A and B.
  3. Flush the column with 5 column volumes of ddH2O using a Manual Run.
  4. Move the input lines from ddH2O into 20% EtOH.
  5. Flush the column with 5 column volumes of 20% EtOH using a Manual Run.
  6. Remove the column.
  7. Turn off the lamp, Parameters –> Lamp (OFF)
  8. Refill the fraction collector racks.
  9. Empty the waste.
  10. Close the laptop lid.

General Tips

  • Make sure to assemble injection loop properly (i.e. check to make sure that the plunger is in the correct orientation,  that the in and out lines are connected to the right valve ports, and that all line attachments are tightened securely).
  • Set the injection volume to slightly less than the full sample volume. This will help to avoid pushing the plunger too far and pushing bubbles through the sample lines.
  • To flow buffer, water, etc. through a column; select a manual run setting and adjust the flow rate and pressure limits appropriately. (For most HiTrap columns, the max pressure is 0.5 MPa and the max flow rate is 5 mL/minute).
  • Always remember to wash the column before and after use and to store it in the appropriate buffer.
  • Be sure to turn the lamp off after use.
  • To find a saved method (i.e. for HIC purification) go to the method templates and select the appropriate method. From the method menu you can then change whatever parameters are necessary such as elution volume, flow rate, and the pressure limit.

Columns

Hydrophobic Interaction Chromatography (HIC) Purification:

  • Max flow: 5 mL/min; Run flow: 1 mL/min; Max pressure: 0.5 MPa (0.3 MPa column + 0.2 MPa back pressure).  
  • Use a system wash method to wash pumps (select from list of application templates)
  • Wash column with ~50mL filtered H2O (scroll through menu and use manual run to flow water through column at  1mL/min, with a pressure limit of 0.5MPa)
  • Equilibrate stacked HIC columns with buffer A (use manual run to flow ~50mL buffer through column)
  • Load sample via super-loop (attached to injection valve)
  • Use a saved affinity purification method. (Set sample injection volume to whatever is applicable. Do a wash with ~100mL buffer A. Then do a step elution into 50-100mL buffer B, collecting 2mL fractions

Ion Exchange Chromatography (IEC) Purification:

  • Max flow: 5 mL/min; Run flow: 1 mL/min; Max pressure: 0.5 MPa (0.3 MPa column + 0.2 MPa back pressure).
  • Use a system wash method to wash pumps
  • Wash column with ~50mL filtered H2O (use manual run to flow water through column at ~1mL/min, with a pressure limit of 0.5MPa)
  • Equilibrate stacked DEAE columns with appropriate buffer (use manual run to flow ~50mL buffer through column)
  • Load sample via super-loop (attached to injection valve)
  • Use a saved IEC purification method. (Set sample injection volume to whatever is applicable. Do a wash with ~100mL low salt buffer. Then elute with a 100mL gradient to high salt buffer, collecting 2mL fractions. To avoid losing protein that does not stick well to column, collect 5mL fractions of wash flow-through (the machine will automatically start collecting 2mL fractions when the elution gradient begins).

Size Exclusion:

  • We currently have a HiPrep 16/60 200 HR size exclusion column (documentation here).  Max flow is 1.0 mL/min.  Pressure limit is 0.35 MPa (0.15 MPa column limit + 0.2 MPa back pressure).  
  • Equilibrate the column with 120 mL ddH20, 240 mL buffer @ 0.3 mL/min, 0.35 MPa pressure limit.  (The lower flow rate it to prevent an overpressure at the ddH2O/EtOH interface.  After the first ~1 hr, you can raise to 0.5 mL/min without issue).  You can use the affinity step gradient program, with line A in ddH2o and line B in your buffer to do this overnight.
  • Dialyze your protein (< 5 mL) into the same buffer you use to equilibrate the column.  (I usually make up ~5 L of dialysis buffer, then filter 1 L of it for use in the column). 
  • Install the 5 mL sample loop and flush it with ~20 mL buffer.  
  • Inject your protein into the sample loop.  I usually load ~4 mL and chase it with 1 mL to make sure the input line is clear.
  • Do a run 120 mL @ 0.5 mL/min, 0.35 MPa pressure limit, collecting fractions ever 2 mL.  
  • When finished with the column, run 120 mL of ddH20, followed by 240 mL of 20% EtOH over the column @ 0.3 mL/min, 0.35 MPa limit.