AMPK Methods and Protocols

9. Wash FLAG monoclonal antibody-coupled affinity resin exten-
   sively in insect lysis buffer (5
   10 volumes).


10. Elute CaMKK2 in insect elution buffer. Aliquot and flash
    freeze eluted protein in liquid nitrogen and store at 80 C.

3.3 Crystallization
for ADaM Site
Activators

1. Dilute full-length phosphorylated α 2 β 1 γ1 to 4 mg/ml
   (30μM) in storage buffer containing a threefold molar excess
   of AMP (90μM), an equimolar (30μM) of staurosporine and
   A-769662 (seeNote 6), and two- to fivefold molar excess
   (60–150μM) of synthetic peptide S108tide or SAMS (see
   Note 7), and incubate on ice for 30 min.


2. Set up hanging drop crystallization experiments in a 24-well
   crystallization plate by adding 500μl of reservoir solution to
   the crystallization plate. We suggest altering the concentration
   of both cocamidopropyl betaine (seeNote 8) and PEG 3350 in
   a grid screen.


3. Add 1.0μl of the AMPK/compound mixture to the reservoir
   solution at a 1:1 ratio, and incubate the crystallization plate at
   4 C. Crystallization experiments can be set at room tempera-
   ture or at 4C. Crystals should appear after 2–4 days, growing
   to their full size within 2 weeks (Fig.2).


4. Crystal size can be increased by one of two methods, either
   protein feeding (seeNote 9) or macro-seeding (seeNote 10).

3.4 Crystallization
for C2-Site Activators

It is possible to use different protein constructs to visualize theγ1-

subunit activator C2, full-length phosphorylatedα 2 β 1 γ1, and full-

length phosphorylated AMPKα1/α2 RIM chimera (seeNote 11).

1. Dilute full-length phosphorylatedα 2 β 1 γ1 as described instep
   1 in Subheading3.3 with the following exceptions: make two
   separate AMPK protein stocks at 4 mg/ml, one containing
   twofold molar excess of AMP (60μM) and the other contain-
   ing twofold molar excess of C2 (60μM), without AMP. Incu-
   bate the protein/compound mixture at 4C for 30 min, and
   then combine prior to setting the hanging drop experiment
   described insteps 2and 3 in Subheading3.3.


2. Dilute full-length phosphorylated AMPKα1/α2 RIM chimera
   [12] (4 mg/ml, 30μM) as described instep 1in Subheading
   3.3 with the following exception exchange AMP for a sixfold
   molar excess of C2 (180μM).


3. Hanging drop crystallization experiments were set up as
   described insteps 2and 3 in Subheading3.3.

Visualization of Drug Binding Sites 23