Protein Expression and Purification Series - Bio-Rad

CHAPTER 7
BIOLOGIC LP SYSTEM
PROTOCOL
Protein Expression and Purification Series
Priming with buffer A and buffer B
1.
2.
A
D
Figure 7.7. Installing a handpacked column with a flow adaptor. A) A syringe filled with
10 ml of water is connected to the tubing on the flow adaptor via the luer lock fittings. 5
ml of water is flowed gently over the column into a waste beaker. This serves to fill up the
tubing with water and provide a first rinse of the resin with water to remove storage buffer.
B) The column inlet and column outlet tubing of the BioLogic LP system are separated for
installation of the column. C) The column outlet tubing of the BioLogic LP system is attached
to the outlet luer lock fitting on the column. 3 ml more water is gently pushed through the
column using the syringe still attached to the flow adaptor tubing. This water serves to
further rinse the resin as well as to purge air bubbles out of the column oulet tubing. D) The
syringe is removed from the flow adaptor tubing and the column inlet tubing that comes
from the MV-6 injection valve is connected to the column putting the column inline for flow
of buffers from the BioLogic LP system.
Using the system’s manual mode operation of the pump, set a flow rate that is safe for the column,
which in this case is 2 ml/min. To do this follow the steps below:
a. From the front panel of the BioLogic LP system, press the Manual mode key, followed by the
Pump instrument key. The yellow light above each key should be on.
b. Using the softkeys below the front panel’s display, press the FLOW softkey.
c. Using the front panel’s keypad, enter a flow rate of 2 ml/min, then press the OK softkey to
accept.
d. Using the softkeys below the front panel’s display, press the BUFFER softkey.
e. Using the Previous and Next keys, toggle until B is bracketed and then press the OK softkey to
select Buffer B.
Place the inlet tubing for Buffer A in the bottle of Buffer A and the inlet tubing for Buffer B in the bottle
of Buffer B. Make sure the tubing goes to the bottom of the bottles.
3. Press the START softkey to begin flow and to purge air that may have entered the system when
the column was connected. Prime the Buffer B inlet tubing with Buffer B and do a final wash of the
column with Buffer B. Allow the system to run for seven minutes (14 ml of Buffer B) then press the
STOP softkey.
B
144 Chapter 7: Purification Protocol for BioLogic LP System
C