3.3 Purification
of AMPK Protein
This is a 3-day process including some longer 8–9 h days in the
laboratory, assuming everything progresses smoothly (Fig.1).
Day 1 AM:
- Equilibrate the Superdex 200 size exclusion chromatography
column with gel filtration buffer at flow rate of 1 mL/min.
Ensure the pressure alarm is set. Equilibration is complete once
a buffer volume greater than 120 mL has passed through the
column. - Remove the column and seal it and keep for day 1 pm below.
Day 1 PM:
- Break open the cell pellet and remove the cell debris from the
solution fraction containing the AMPK protein. To do this first
thaw the frozen cell pellets on ice and resuspend in lysis buffer.
Use 4–5 mL of buffer per gram wet weight of cells. - Lyse the cells on ice using sonication in an appropriately sized
beaker. Insert the sonicator probe about halfway down the
depth of the lysate. In our hands cells are broken by sonicating
on ice at (on 1 s, off 1 s) 40% power for a total time of 1 min
and 30 s. However, these parameters will vary dependent on
specific equipment, so it is critical to be certain that cells are
broken but that material is not over-sonicated as this will heat
and damage the sample. Two indicators of cell breakage are the
change in the visible appearance of the suspension and the size
of the remaining pellet after the next centrifugation step. - Pour the lysate into 50 mL centrifuge tubes and centrifuge at
20,000 rpm (33,000g) J-26 XP centrifuge for 45 min at
4 C. The supernatant should be opaque, and the pellet which
200
116.3
97.4
66.3
55.4
36.5
31
21.5
14.4
6
3.5
Express and Harvest
Lysis by sonication
Size Exclusion Chromatography
Pooled
Ni Peak Size Exclusion Chromatography
ab
AV I - β
His-α
γ
Fig. 1Purification of AMPKα 1 β 2 γ3. (a) Purification scheme. (b) SDS-PAGE analysis of HisTrap purified AMPK
complex and the analysis of size exclusion chromatography peak
AMPK Crystallisation 7