Protein Expression and Purification Series - Bio-Rad

CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL
Protein Expression and Purification Series
Figure 8.22. Fraction Collection Scheme programming. This window allows programming of the BioFrac fraction collector. The
options are: Collect All, where all fractions are collected and each has the same volume; Threshold, where fractions are collected
if the absorbance is greater than a set value; Collection Windows, where fractions of differing volume are collected in different time
frames (windows); and Threshhold & Collection Windows, where fractions collected during certain time frames have an absorbance
greater than a set value. Collection Windows will be used for the GST-DHFR-His protocol. Larger volume fractions will be collected
for proteins that do not bind to the column (flowthrough fraction) and are washed off the column (wash fractions) while smaller
fractions will be collected when the GST-DHFR-His elutes. This allows for more concentrated GST-DHFR-His samples.
3.
4.
5.
Program the first window by typing 6.00 into the Start (ml) box, 12.00 into the End (ml) box and 4.00
into the Frac. Size (ml) box. Press the Save Window button.
Table 8.1 Programming BioFrac fraction collector collection windows.
Step Number Start (ml) End (ml) Frac. Size (ml) Product being collected
1. 6.00 12.00 4.00 Flowthrough
2. 12.00 23.00 4.00 Wash
3. 23.00 37.00 1.00 Eluted GST-DHFR-His
Press the Add Window button. Enter 12.00 for the Start (ml), 23.00 for the End (ml) and 4.00 for Frac.
Size (ml). Press the Save Window button.
Press the Add Window button. Enter 23.00 for the Start (ml), 37.00 for End (ml) and 1.00 for Frac. Size
(ml). Press the Save Window button, then Close.
Programming the pump and UV lamp steps of the protocol
1. Program the remaining steps using the Add Step icons on the left side of the screen. Note that Step
1 has already been added to your protocol since you already programmed the fraction collector. Table
8.2 on page 182 includes all of the remaining steps that need to be programmed. Examples of how to
program Steps 2 and 3 are outlined here.
Note: The software adds a step above whatever line is shaded in blue. For example, in Figure 8.23,
if a step were to be added with the Collection Fractions step highlighted, the step would be added
before this step. To add a step after Collection Fractions, have the End of Protocol step highlighted.
180 Chapter 8: Purification Protocol for BioLogic DuoFlow System