Protein Expression and Purification Series - Bio-Rad

CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL
Protein Expression and Purification Series
Section 2. Affinity Chromatography using Native IMAC Protocol to Purify GST-
DHFR-His
Manually preparing the DuoFlow system and IMAC Ni-charged column
1. Prime the Pump Inlet tubing with Buffer A and Buffer B
The inlet tubing is currently filled with water. At this point, the inlet tubing will be primed with Buffer A and
Buffer B in preparation for cleaning and equilibrating the column.
1.
2.
3.
4.
5.
Ensure the pumps are stopped and the AVR7-3 inject valve is in the Load (L) position on the Manual
screen.
Place the tubing for Pump A into the container of Buffer A. Make sure that the tubing sits near the
bottom of the container. Tape the tubing down if necessary.
Place the tubing for Pump B into the container of Buffer B. Make sure that the tubing sits near the
bottom of the container. Tape the tubing down if necessary.
Attach the 10 ml syringe to the priming port for Pump A, turn the priming port counterclockwise to
open, and pull 1–2 ml of Buffer A into the syringe to remove any air bubbles introduced into the Inlet
line. Turn the priming port clockwise to close and remove the syringe. Expel the buffer in the syringe
into the waste beaker.
Repeat step 4 for priming port B.
2. Cleaning and equilibrating the IMAC Ni-charged column in Manual mode
At this point, the lines throughout the entire system will first be filled with Buffer B (20 mM sodium
phosphate buffer, 300 mM NaCl, 250 mM imidazole, pH 8) to wash any remaining contaminants off of the
IMAC column. Then the lines will be filled with 2% Buffer B (20 mM sodium phosphate buffer, 300 mM
NaCl, 5 mM imidazole) to flush the 100% Buffer B solution out of the system and replace it with 2% Buffer
B (the same composition buffer that the soluble fraction is in). The column will be equilibrated at this point
and ready for use.
1.
2.
Set the AVR7-3 inject valve to position L (Load).
Set the Gradient Pump Flowrate to 2.00 ml/min, Inlet B to 100%, High limit to 200 psi and Low limit to
0 psi, then press the Start button (see Figure 8.12). Allow the wash to proceed for five minutes. While
the pump is running, continue to steps 4–6.
Figure 8.12. Manual wash. The column and tubing are first washed with 100%
Buffer B at 2.00 ml/min for five minutes. In chromatography nomenclature, this
is a 10 CV (column volume) wash since 10 ml of buffer are flowed over a 1 ml
column. This step washed off any storage buffer remaining on the column and
any residue from previous purifications (if present).
174 Chapter 8: Purification Protocol for BioLogic DuoFlow System