Protein Expression and Purification Series - Bio-Rad

CHAPTER 4
11 ml CULTURE
PROTOCOL
Protein Expression and Purification Series
Centrifugation Purification Workflow
Affinity and Size Exclusion (Desalting) Purification of GST-DHFR-His (continued)
Desalting of the GST-DHFR-His protein (Eluate fraction) to remove
imidazole. Prepare desalting column (with green cap) by inverting
sharply several times to resuspend gel.
Snap off bottom tab and place in 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, then place column in clean 2 ml tube.
Centrifuge for two minutes at 1,000 x g. Discard remaining
packing buffer and collection tube.
Label new 2 ml tube Desalted Eluate. Carefully apply 75 µl of Eluate
fraction directly to the center of column. Be careful not to touch resin
with pipet tip.
Centrifuge for four minutes at 1,000 x g. Repeat addition of 75 µl of
Eluate fraction to column and centrifugation.
After second spin, discard column. You should now have ~150 µl
of Desalted eluate (GST-DHFR-His).
Desalted
eluate
Spin 2 min at 1,000 x g
+ 75 µl
Eluate
Spin 4 min at 1,000 x g
114 Chapter 4: 11 ml Culture Protocol
for Centrifugation Purification
X 2