Protein Expression and Purification Series - Bio-Rad

CHAPTER 3A
ADVANCE PREP
CENTRIFUGATION PROCES
Protein Expression and Purification Series
5. Aliquot 200 µl of sterile water into a 2 ml microcentrifuge tube for each workstation.
6. Set up student workstations according to the student workstation list below.
Note: The Profinity IMAC Ni-charged resin is supplied as a 50% slurry in a 20% ethanol solution. Make
sure to thoroughly resuspend the resin by vortexing and aliquot 250 µl of the resuspended slurry into
microcentrifuge tubes for the student workstations. This is equivalent to 125 µl of resin per student
workstation, and the students will only use 100 µl of resin.
Student Workstations
Each student team requires the following items to purify their GST-DHFR-His samples using affinity
chromatography followed by size exclusion chromatography (desalting).
Material Needed for Each Workstation Quantity
Soluble lysate fraction 600–750 µl
Profinity IMAC Ni-charged resin slurry 250 µl slurry
Distilled water 200 µl
Equilibration buffer 500 µl
Lysis buffer 2/Wash buffer 600µl
Elution buffer 400 µl
Empty Micro Bio-Spin column with cap and yellow tip closure 1
Desalting column, with green cap* 1
Microcentrifuge tubes, 2 ml 6
Screwcap microcentrifuge tubes, 1.5 ml 4
Laemmli sample buffer (leftover from previous activity) 1 ml
2–20 µl adjustable-volume micropipet and tips 1
20–200 µl adjustable-volume micropipet and tips 1
100–1,000 µl adjustable-volume micropipet and tips 1
Marking pen 1
*Note: to avoid confustion between the desalting column and the empty column, you may choose to wait
to provide the desalting column until after the affinity purification step has been completed.
Common Workstation Quantity
Tube roller or mini rocker 1
Water bath or dry bath set to 95°C 1
Microcentrifuge with variable speed setting >16,000 x g 1
66 Chapter 3A: Advance Preparation
for Centrifugation Protocols