Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Figure 8.28. Typical trace of purification of GST-DHFR-His. The left axis has been set to a maximum value of 2.00 AU and the
right axis is set to a maximum value of 50 mS/cm. Trace 1 is the UV trace. It increases after the sample has been loaded, and all
proteins that do not bind (the flowthrough) flow past the column and through the detectors. The UV curve then decreases to baseline
(close to 0.0) as the column is washed, and all non-binding proteins are washed off. The % Buffer B can be seen by looking at
curve 3. Once % Buffer B goes to 100%, the UV curve increases again as the GST-DHFR-His elutes. The UV curve then decreases
as all of the GST-DHFR-His washes off but does not drop to baseline due to the 250 mM imidazole in 100% Buffer B absorbing at
280 nm. Finally, when % Buffer B drops to zero, the UV curve drops back to baseline values. Curve 2 is the conductivity plot. The
conductivity drops slightly when the flowthrough passes through the conductivity detector. Once all the non-binding protein passes,
the conductivity goes back to its initial set point. Once % Buffer B increases to 100%, the conductivity increases slightly due to the
presence of 250 mM imidazole and then drops back to its baseline when % Buffer B goes to 0%.
bottom right of the status bar.
8. Figure 8.28 shows a typical chromatogram for this separation.
9.
Label your fractions numerically (1, 2, 3, etc.) making sure to follow the serpentine path that the
fraction collector used.
Note: The BioFrac fraction collector automatically skips tube 1. Tube 2 will be the first one with liquid
in it.
10. Cover your fractions with Parafilm and store at 4°C until you are ready to desalt and analyze them via
SDS-PAGE and enzyme activity.
Cleaning the BioLogic DuoFlow System After Use
In order to keep any instrument in good running form, it is critical to perform certain tasks to make sure
that the instrument is clean and ready for use by the next person. For the BioLogic DuoFlow system, it is
important to remove all salt-containing solutions from the lines and all bacterial lysate components. The
salts can precipitate out over time clogging fittings, tubing, and flow cells. Bacterial lysates can degrade
resulting in growth of molds and mildews that also will contaminate and clog fittings, tubing, and flow cells.
1.
2.
3.
4.
5.
Remove your column but leave the red adaptor fittings attached to the instrument fittings.
Place the luer lock closure column fittings on the inlet and outlet of the column to keep the column
from drying out when stored.
Connect the column inlet and outlet tubing by connecting the two red fittings adaptors still connected
to the tubing.
Place the buffer A and buffer B inlet lines into a beaker containing 200 ml of high-quality water.
In manual mode, make sure that the AVR7-3 valve is in the Load (L) position.
Chapter 8: Purification Protocol for BioLogic DuoFlow System
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CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL