Protein Expression and Purification Series - Bio-Rad

CHAPTER 7
BIOLOGIC LP SYSTEM
PROTOCOL
Protein Expression and Purification Series
Preparing SDS-PAGE samples of flowthrough, wash and eluted GST-DHFR-His fractions
1.
2.
3.
4.
5.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of
your flowthrough fraction and label the tube “Flowthrough PAGE” with your initials.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of
your wash fraction and label the tube “Wash PAGE” with your initials.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of
your first eluted GST-DHFR-His fraction and label the tube “Fraction A PAGE” with your initials.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of
your second eluted GST-DHFR-His fraction and label the tube “Fraction B PAGE” with your initials.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of
your third eluted GST-DHFR-His fraction and label the tube “Fraction C PAGE” with your initials.
6. Heat the five SDS-PAGE samples at 95°C
for five minutes.
7. Store the SDS-PAGE samples at –20°C until they are ready to analyze via SDS-PAGE.
Desalting your three eluted GST-DHFR-His fractions
1.
2.
3.
4.
5.
Label three Micro Bio-Spin desalting columns A, B, and C with your initials. These fractions will
correspond to the three fractions you chose to be the eluted GST-DHFR-His fractions from your
chromatogram:
Fraction A = First eluted GST-DHFR-His fraction
Fraction B = Second eluted GST-DHFR-His fraction
Fraction C = Third eluted GST-DHFR-His fraction
Invert three desalting columns (with green caps) sharply several times to resuspend the settled gel and
remove any bubbles. The resin should settle into the column, and little to no resin should remain in the
green cap.
Snap off the bottom tab of the column and place the column into a clean 2 ml microcentrifuge tube.
Remove the columns’ green caps. If the columns do not begin to flow, push the caps back on the
columns and then remove them again to start the flow.
Allow the excess packing buffer to drain by gravity to the top of the gel bed (about two minutes), then
place the three columns into three clean 2 ml microcentrifuge tubes.
6. Centrifuge the three columns for two minutes in a microcentrifuge at 1,000 x g (See Appendix C for
more information on setting centrifugation speeds) to remove the remaining packing buffer. Discard
the buffer and the three microcentrifuge tubes. Keep the three columns for the following steps, being
careful not to disturb the resin.
7. Label three clean 2 ml microcentrifuge tubes “ Desalted A,” “Desalted B” and “Desalted C” and
each with your initials, and place the columns into the corresponding 2 ml micocentrifuge tubes.
Carefully apply 75 µl of each of your eluted fractions chosen in step 1 directly to the center of the
column. Be careful not to touch the resin with the pipet tip.
8.
After loading the samples, centrifuge the column for four minutes at 1,000 x g.
154 Chapter 7: Purification Protocol for BioLogic LP System