Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Purification Workflow for BioLogic DuoFlow 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 two 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 minutew 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 ~150ul of
desalted GST-DHFR-His for each elution fraction.
Chapter 8: Purification Protocol for BioLogic DuoFlow System
197
Desalted
A, B or C
2 min at 1,000 x g
+ 75 µl
eluate
4 min at 1,000 x g
X 2
CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL