AMPK Methods and Protocols

3. Wash anti-FLAG M2 affinity agarose gel three times by resus-
   pension in 1 ml of FLAG agarose wash buffer, followed by
   centrifugation at 3,350gfor 3 min.


4. After the final centrifugation step, resuspend anti-FLAG M2
   affinity agarose gel in CaMKK2 expression cell lysate. Place on a
   rotating wheel at 4C for 1 h, and pellet anti-FLAG M2 affinity
   agarose gel by centrifugation at 3,350gfor 3 min.


5. Wash anti-FLAG M2 affinity agarose gel three times by resus-
   pension in 1 ml of FLAG agarose wash buffer, followed by
   centrifugation at 3,350gfor 3 min.


6. For elution, after the final centrifugation step, resuspend in
   FLAG agarose elution buffer (10μl buffer/10μl agarose),
   and place on rotating wheel at 4C for 3 h. Pellet agarose by
   centrifugation at 3,350gfor 3 min, and repeat the 3 h
   elution step. Combine CaMKK2 elutions and dispense into
   20 μl aliquots. Flash freeze aliquots in liquid N 2 for long-
   term storage at
   80 C.


7. For Thr-172 phosphorylation by CaMKK2, immobilize
   AMPK on glutathione agarose (seeNote 12), and wash three
   times in glutathione agarose wash buffer, as described instep 1
   in Subheading3.2. Uniformly resuspend glutathione agarose
   in an equal volume of CaMKK2 phosphorylation buffer (room
   temperature), and add CaMKK2 (20 ng enzyme/10 μl
   agarose).


8. Place on a rotating wheel at room temperature for 10 min, and
   pellet glutathione agarose by centrifugation at 3,350gfor
   3 min.


9. Wash glutathione agarose three times by resuspension in 1 ml
   of glutathione agarose wash buffer, followed by centrifugation
   at 3,350gfor 3 min.


10. For elution, after the final centrifugation step, resuspend in
    glutathione agarose elution buffer (10μl buffer/10μl aga-
    rose), and place on rotating wheel at 4C for 5 min. Pellet
    glutathione agarose by centrifugation at 3,350gfor 3 min.
    Dispense AMPK elution into 50μl aliquots (seeNotes 14and
    15 ). Flash freeze aliquots in liquid N 2 for long-term storage at
    
    80 C.


11. If desired, for Thr-172 dephosphorylation, immobilize AMPK
    on glutathione agarose, and wash three times in glutathione
    agarose wash buffer, as described insteps 1– 4 in Subheading
    3.2.


12. Uniformly resuspend glutathione agarose in an equal volume
    ofλ-phosphatase dephosphorylation buffer (room tempera-
    ture), and addλ-phosphatase (200 activity units/10μl aga-
    rose). Place on rotating wheel at room temperature for 30 min.

166 Jonathan S. Oakhill et al.