- Having decided how much CaMKK2 to use to obtain maximal
Thr172 phosphorylation, scale up the activation assay with the
required amount of CaMKK2—typically 5 mg of purified
AMPK is phosphorylated. After the scaled-up assay, the mix-
ture is applied to a HiLoad 16/600 Superdex 200 pg gel
filtration column with an in-line 5 mL glutathione-Sepharose
FF column, both pre-equilibrated in gel filtration equilibration
buffer run at 1 mL/min (seeNote 8). - Fractions containing the desired protein (usually monitored by
SDS-PAGE) are pooled, concentrated using centrifugal con-
centrators (following manufacturers’ instructions) to3 mL,
dialyzed against 41 L of phosphorylated AMPK dialysis
buffer for 30 min each (seeNote 9), and stored at 20 C.
3.4 Standard
In-Solution Kinase
Assay
This protocol refers to our standard final assay volume of 25μL.- Mix the following components in 1.5 mL microcentrifuge
tubes: (1) 5μL of peptide substrate (SAMSorAMARA, final
concentration 200μM); (2) 5μL of AMP (final concentration
200 μM); (3) 5μL of AMPK (appropriately diluted to ensure
activity is proportional to amount added); (4) 5μL of kinase
assay buffer. - Start the reaction (typically at 15 s intervals) by adding 5μLof
MgCl 2 /[γ-^32 P]ATP (final concentration 5 mM/200μM), vor-
tex, and incubate at 30C for 10 min. Also perform blanks
where the peptide is omitted. It is also possible to prepare
master mixes that include MgCl 2 /[γ-^32 P]ATP, in which case
the reaction is initiated by addition of AMPK instead. AMP can
also be replaced in the master mix with assay buffer to deter-
mine AMPK activity in the absence of allosteric activator or
replaced by another allosteric activator such as A769662. - Stop the reactions at 15 s intervals by removing 15μL and
pipetting onto a P81 paper square held with forceps. As soon as
the fluid has soaked in (about 1 s), drop the square into a large
beaker containing 500 mL of 1% phosphoric acid. - Once all the reactions have been stopped, stir the beaker con-
taining the paper squares gently on a magnetic stirrer for 5 min
at room temperature. - Pour off the phosphoric acid to radioactive waste. Add 500 mL
of 1% phosphoric acid and again stir the beaker containing the
paper squares gently on a magnetic stirrer for 5 min at room
temperature. Pour off the phosphoric acid to radioactive waste
and repeat this washing step once more. - Lay filters out on absorbent paper on a radioactive spill tray and
allow to dry in the air.
Cell-Free Assays for Regulatory Ligands 77