AMPK Methods and Protocols

3.2 Validation
of Putative AMPK
Targets

All procedures are to be performed at room temperature unless

otherwise specified.

1. Prepare a fresh SDS-PAGE gel for each putative target (see
   Note 13). For each target protein of interest, prepare two
   reaction tubes: one with and one without AMPK. For reactions
   containing AMPK, prepare Buffer B by adding 1μl of recom-
   binant AMPK to 3μl of Buffer A (seeNote 4).


2. In a clean centrifuge tube, prepare Buffer C by adding 1μlof
   250 mM MgCl2,0.4μlof[γ-^32 P] ATP, 5μl of 10 mM AMP,
   and 3.6μl of Buffer A (seeNote 4).


3. On ice, add 10μl of Buffer C to each reaction tube, 1μgof
   target substrate, 4μl of Buffer B for reactions containing
   AMPK, and bring the reaction volume to 50μl with Buffer A
   (seeNotes 14– 16 ).


4. Incubate reactions at 37C for 1 h (seeNote 17).


5. Stop reactions with the addition of 16.7μlof4Laemmli
   buffer, and place in the heating block set at 100C for 15 min.


6. While the reactions are incubated, set up SDS-PAGE electro-
   phoresis equipment per the manufacturer’s instructions, and
   fill the inner and outer buffer chambers with Tris-glycine run-
   ning buffer [7, 8].


7. Load equal volumes of each sample into individual wells, set the
   power supply to 100 volts, and operate until the bromophenol
   reaches the bottom of the gel.


8. Remove the gel from the electrophoresis cassette, immerse it in
   Coomassie stain, and gently agitate with the orbital shaker or
   rocker for 15 min or until the Coomassie stain has completely
   penetrated the gel.


9. Place the gel in destaining solution and gently agitate. Periodi-
   cally change the destaining solution until the gel regains its
   transparent appearance and the target protein bands are clearly
   visible. Note that under the reaction conditions present in this
   protocol, AMPK subunit concentrations are below the detect-
   able limit of Coomassie stain. At this point, a Geiger counter
   can be used to assess the level of radiation in the gel. Be sure the
   gel is not submerged in destaining solution while assessing the
   radioactivity, because water will shield radiation causing a false
   reading (Fig.1).


10. Lay a clean transparency sheet on a bench or flat surface.
    Dampen a piece of Whatman filter paper with water and lay it
    on top of the transparency sheet. Gently remove the gel from
    destaining solution and place it on top of the Whatman paper.
    Once the gel is in place and flat on the Whatman paper, lay a

104 Brendan Gongol et al.