Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
1.5 Flush the system through with water to the fraction collector
Up to this point, the inlet tubing for Pump A and Pump B,
as well as all the tubing up to the AVR7-3 valve, have been
flushed with water. Now it is time to flush the entire system
beyond the AVR7-3 valve (the column, UV and conductivity
detectors and fraction collector and all associated tubing)
with water as well.
1.
2.
3.
On the Manual screen, move the AVR7-3 valve back
to position L (Load) by clicking “L” (Figure 8.4A). Verify
that L is now displayed on the AVR7-3 valve.
From the gradient pump control panel on the Manual
screen, set the pump flow rate to 2.0 ml/min, set the
composition to 50% Buffer B and start the pump
(Figure 8.6). Make sure that you have at least 200 ml of
water remaining in your beaker and that both the inlet
lines are at the bottom of the beaker.
In the Fraction Collector window on the Manual screen
ensure that the System button is selected (Figure 8.7).
1.6 Turn on the UV lamp
Check that the lamp is on by looking at the UV Detector
panel (Figure 8.8); if it is not, press the ON button. The light bulb icon should be yellow when the UV lamp
is on.
1.7 Manual screen chromatogram window
A feature of the Manual screen is its ability to display up to eight traces on a chromatogram—including
UV/Vis, pH, conductivity, % Buffer B and pressure traces—over a 10-minute interval. This is useful during
column equilibration. The chromatogram window is displayed at the bottom of the screen under the valve
control panel (Figure 8.9).
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Chapter 8: Purification Protocol for BioLogic DuoFlow System
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Figure 8.7. Manual DuoFlow system operation fraction
collector control. The BioFrac fraction collector has two
operating modes: System—controlled by the DuoFlow
system and Local—controlled from its own faceplate in
stand-alone mode. When in System mode, the fraction
collector control panel will show fields for Rack type,
Start tube, End tube, Fraction size, Tube number, Volume
left, as well as a toggle button for Start and Stop, and a
button for Advance.
Figure 8.8. Manual control of the UV detector. The UV lamp automatically turns on
when you turn on power to the workstation. The UV lamp can be turned on and off
by clicking the On and Off buttons from the UV detector control panel on the Manual
screen.
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Figure 8.9. Manual screen chromatogram. Features of the chromatogram window include: 1. The time axis is reset automatically
at the end of 10 minutes or reset manually by clicking the Clear Traces button. 2. The chromatogram window can be enlarged by
pressing the Resize button. 3. Which chromatogram trace is to be displayed can be selected by using the drop-down menus on the
upper right and left of the display 4. The Y-axis scale can be changed using the scroll bars on the right or left of the display 5. The
maximum and minimum axis settings can be changed by pressing Settings on the Manual screen toolbar (see Figure 8.2).
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CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL