AMPK Methods and Protocols

including blind samples (seestep 6) and three consecutive IPs

for measuring the activities ofα 2 β 2 γ3,α 2 β 2 γ1, andα 1 β 2 γ 1

(in that order).

2. Make up a fresh batch of IP-buffer and keep it on ice or at 4C
   (seeNote 6).


3. Choose the appropriate Protein A or G agarose (agarose beads)
   according to the antibody to be used in the IP (seeNote 7).


4. Wash the agarose beads thrice in IP-buffer and make up a 50:50
   slurry suspension (seeNote 2).


5. Place the PCR-plate in the aluminum block on ice and add
   20 μl of agarose beads (50:50 slurry) to each well in the
   PCR-plate (one well per sample).


6. Prepare to set up a number (e.g., triplicate) of “blind” samples
   that could be a lysate pool of the samples analyzed. These blind
   samples must be used as background activity and are subtracted
   from the other samples in the calculations at the end of the
   assay. Set up the blind samples as the rest of the samples, but do
   not add antibody (or choose an irrelevant antibody).


7. Add IP-buffer according to your calculations. The total volume
   of the IPs should be 200μl, so the IP-buffer should be 200μl
   minus 20μl of agarose (50:50 slurry), minus 10μl of antibody,
   and minusxμl of lysate (seestep 9).


8. Add 10μl of AMPKγ3 antibody to each IP (seeNote 8) and
   instead 10μl of IP-buffer to the blind samples.


9. Addxμl of lysate (seeNote 6). If assaying the activity of three
   heterotrimeric complexes with three consecutive IPs, we rec-
   ommend to IP on at least 250μg of lysate.


10. Put on the silicone sealing mat and secure it to the PCR-plate
    in the “sandwich device” (Fig.1B).


11. Let the IPs rotate end over end at 4C overnight (seeNote 9).


12. On Day 2, set up a new PCR-plate like the day before. Add
    20 μl of Protein G agarose and 10μl of AMPKα2 antibody to
    the IPs and Protein G agarose and 10μl of IP-buffer to the
    blind samples.


13. Release the “γ3” PCR-plate from the “sandwich device” after
    rotation and spin down the agarose for 1 min at 500gand
    4 C. Place the PCR-plate on ice (in the 96-well aluminum
    block).


14. Using an eight-channel pipette, transfer 170μl of the superna-
    tant from each well to the new “α2” PCR-plate (seeNote 10).
    This is the most difficult part of the assay. Be careful not to
    transfer any agarose (seeNote 11).

220 Jesper B. Birk and Jørgen F. P. Wojtaszewski