Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Removing Imidazole (Desalting) from the Purified GST-DHFR-
His and Preparing Samples for SDS-PAGE Analysis
Student Workstations
Each student team requires the following items to desalt three of their fractions in which they think their
GST-DHFR-His samples reside and to prepare SDS-PAGE samples:
Material Needed for Each Workstation Quantity
Chromatogram from GST-DHFR-His purification 1
Fractions from GST-DHFR-His purification varies
Desalting column 3
Screwcap microcentrifuge tubes, 1.5 ml 6
Laemmli buffer (left over from previous activity) 1 ml
Microcentrifuge tubes, 2 ml 6–12
20–200 µl adjustable-volume micropipet and tips 1
Marking pen 1
Common Workstation Quantity
Microcentrifuge with variable speed setting >16,000 x g 1
Dry bath or water bath set at 95°C 1
At this point, there might be several fractions that contain the eluted GST-DHFR-His protein. It is important
to determine which fractions the purified GST-DHFR-His is present in and which fraction(s) have the highest
concentration of GST-DHFR-His. It might be assumed that the fractions marked by the DuoFlow software
on the chromatogram where the peak eluted are the specific fraction tubes where the protein can be
found. However, it should be noted, that there is a time delay from when the absorbance of the fraction
is measured on the UV detector to when it flows through the tubing past the conductivity meter and out
into the fraction collector. This is called a delay time (or volume). For example, in Figure 8.29, according
to the chromatogram, the protein should have eluted in fractions 7–8. Again, this would be assuming that
there is no delay between when the protein is detected in the UV detector and when it drops into a fraction
collector tube.
Figure 8.29. Chromatogram of a run. If there was no delay volume between the UV detector and fraction collector, the GST-DHFR-
His would be assumed to be in fractions 7, 8, and 9. However, there is a finite delay time between detection and collecting that can
be calculated or measured.
The delay time (volume) can be calculated by measuring the length of tubing present between the detector
and the fraction collector as well as knowing the volume of all fittings in between the UV monitor and the
fraction collector. It can also be measured experimentally ahead of time by attaching a syringe to the fitting
Chapter 8: Purification Protocol for BioLogic DuoFlow System
187
CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL