Protein Expression and Purification Series - Bio-Rad

CHAPTER 7
BIOLOGIC LP SYSTEM
PROTOCOL
Protein Expression and Purification Series
Purification Workflow for BioLogic LP System
Desalting your three eluted GST-DHFR-His fractions
Label three Micro Bio-Spin desalting columns A, B, and C. These
fractions correspond to the three fractions you chose from your
chromatogram.
Prepare desalting columns by inverting sharply several times to
resuspend gel.
Snap off bottom tabs and place each column into a 2 ml
microcentrifuge tube. Remove green top cap. If the column does not
begin to flow, push the cap back on the column and then remove it
again to start the flow.
Allow excess packing buffer to drain by gravity to top of resin
bed. After draining, place columns in clean 2 ml tubes.
Centrifuge for 2 minutes at 1,000 x g. Discard remaining
packing buffer and collection tubes.
Label three clean 2 ml microcentrifuge tubes Desalted A, Desalted B,
Desalted C. Carefully apply 75 µl of each of your eluted
fractions directly to the center of the corresponding column. Be
careful not to touch resin with pipet tip.
Centrifuge for four minutes at 1,000 x g. Carefully apply another
75 µl of each of your eluted fractions to the corresponding column
and centrifuge again.
After second spin, discard columns. You will have ~150 µl
of Desalted GST-DHFR-His for each elution fraction.
Desalted
A, B or C
2 min at 1,000 x g
+ 75 µl
eluate
4 min at 1,000 x g
X 2
162 Chapter 7: Purification Protocol for BioLogic LP System