AMPK Methods and Protocols

followed by 21 mL of IP buffer. Resuspend the final pellet in

1 mL total volume of IP buffer and divide into 20 50 μL

aliquots.

4. To each 50μL aliquot of protein G-Sepharose, add an appro-
   priate amount of cell lysate (as determined by titration in a pilot
   experiment: 100–300μg is a good starting range for most cell
   types). Add 500–700μL of IP buffer to ensure adequate mix-
   ing and then mix for 2 h at 4C on a roller mixer.


5. Centrifuge the mixture at 17,000gfor 3 min at 4C and
   wash the pellet with 11 mL ice-cold IP buffer containing
   0.5 M NaCl, then 11 mL ice-cold IP buffer, and then
   1 1 mL ice-cold assay buffer. Resuspend in a final volume
   of 320μL assay buffer.


6. Divide the mixture into 3 100 μL aliquots in 3 pre-labeled
   microcentrifuge tubes. Centrifuge the mixture at 17,000g
   for 3 min at 4C, remove 80μL of supernatant, and retain the
   pellets on ice for assay.


7. Start the assay reactions at 15 s intervals by adding 30μL of the
   appropriate assay mix (either “þpeptide” or “– peptide”) to
   the 20μL of immunoprecipitate. After each addition, cap the
   tube and insert it into an orbital shaker (operating at about
   1000 rpm, which keeps the Sepharose beads suspended)
   within a benchtop air incubator maintained at 30C.


8. Continue incubations for 15 min at 30C.


9. Stop the reactions at 15 s intervals and wash and count the
   filters as described insteps 4– 10 Subheading3.4.


10. Activities may be presented as phosphate incorporated in
    nmol/min/mg, where “mg” is the amount of protein in the
    original lysate immunoprecipitated. Remember to correct for
    the fact that only 30μL (assuming that was indeed the volume
    sampled) of the 50μL assay had been analyzed.

3.6 Allosteric
Activation by Ligand
Binding the Nucleotide
or ADaM Sites

For allosteric activation assays using AMP, a greater degree of

activation is obtained if assays are carried out in 5 mM ATP com-

pared to the standard conditions of 200μM, possibly because this

concentration of ATP depresses the basal activity [8]. If the con-

centration of ATP utilized is increased, we keep MgCl 2 at a con-

centration 4.8 mM above the concentration of ATP, because with

that protocol the concentration of the Mg.ATP complex varies as a

constant proportion of the total ATP [24] (do not vary MgCl 2 and

ATP in a fixed ratio). If using a higher final ATP concentration, it

may also be necessary to increase the specific radioactivity by adding

more labeled ATP. Since ATP slowly breaks down

non-enzymatically to ADP and/or AMP during the assay, keep

the incubation times short (5–10 min). It is also recommended to

use the SAMS peptide rather than the AMARA peptide as

Cell-Free Assays for Regulatory Ligands 79