AMPK Methods and Protocols

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:

1. 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.


2. Remove the column and seal it and keep for day 1 pm below.

Day 1 PM:

3. 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.


4. 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.


5. 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