Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
4.
Allow the excess packing buffer to drain by gravity to the top of the gel bed (about 2 minutes), then
place the column into a clean 2 ml tube.
5. Centrifuge for 2 minutes in a microcentrifuge at 1,000 x g (see Appendix
C for more information
about setting centrifuge speed) to remove the remaining packing buffer. Discard the buffer and the
microcentrifuge tube. Keep the column for the following steps.
6. Label a clean 2 ml microcentrifuge tube “ Desalted eluate” with your initials and place the column into
the 2 ml microcentrifuge tube. Carefully apply 75 µl of “Eluate” fraction directly to the center of the
column. Be careful not to touch the resin with the pipet tip.
7.
After loading the sample, centrifuge the column for 4 minutes at 1,000 x g.
8. Carefully apply another 75 µl of “Eluate” fraction directly to the center of the column, again being
careful not to touch the resin with the pipet tip.
9.
After loading the sample, centrifuge the column for 4 minutes at 1,000 x g.
10. You should now have approximately 150 µl of desalted eluate in the labeled tube.
11.
Discard the column.
Prepare SDS-PAGE samples
1. In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of
flowthrough and label the tube “Flowthrough PAGE” with your initials.
2.
3.
4.
5.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of
wash fraction and label the tube “Wash PAGE” with your initials.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 25 µl of Laemmli sample buffer with 25 µl of
Eluate and label the tube “Eluate PAGE” with your initials.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 25 µl of Laemmli sample buffer with 25 µl of
desalted eluate and label the tube “Desalted Eluate PAGE” with your initials.
Heat the Soluble PAGE, Flowthrough PAGE, Wash PAGE, Eluate PAGE and Desalted Eluate PAGE
samples at 95°C for 5 minutes.
6. All SDS-PAGE samples can be stored at –20°C until SDS-PAGE analysis is to be performed.
Storage of chromatography fractions
The flowthrough, wash, eluate and desalted eluate fractions should all be stored at 4°C until the DHFR
Enzymatic Assay is to be performed. Do not freeze the samples.
Chapter 4: 11 ml Culture Protocol for
Centrifugation Purification
103
CHAPTER 4
11 ml CULTURE
PROTOCOL