- Add small amounts of the ATP to the buffer, allowing each
addition to fully dissolve before adding more. Maintain the pH
between 7 and 8 using 1 mol.L^1 NaOH, to prevent any
breakdown of ATP to ADP and AMP. - After dissolving all the ATP, make the final volume up to 20 mL
using kinase assay buffer. - The exact concentration of ATP should be calculated, based on
a molar extinction coefficient for ATP of 15,000 at 260 nm.
Dilute a sample of the putative 100 mM ATP solution 5000-
fold in water (serial dilution of 1:100, then 1:50) and monitor
its absorbance at 260 nm, which should be 0.3. - Store unlabeled ATP at 20 C in frozen aliquots; add radio-
active ATP as required, to give a final specific radioactivity of
50–300 cpm/pmol or more if using ATP at higher concentra-
tions in the assay (e.g., final concentrations of 1 or 5 mM,
rather than 200μM,seeSubheading3.6). For a tip regarding
safe handling of radioactive ATP solutions,seeNote 6.
3.2 Expression
and Purification
of AMPK and CaMKK2
from Bacterial Cells
A polycistronic expression plasmid [19] allows expression of all
three subunits within the same bacterial cell, facilitating the forma-
tion and stabilization of the complex. Our AMPK polycistronic
plasmids encoding AMPK [12, 23] produce His-tagged proteins,
while our CaMKK2 plasmids [20] produce GST-tagged proteins;
other tags may also be used.- Transform both plasmids by incubating 0.1–1μg DNA with
25 μL BL21-CodonPlus (DE3)-RIL bacterial cells and incu-
bate on ice for 30 min. Heat shock the cells at 42C for 1 min
followed by a further 5 min on ice. Add 200μL of SOC
medium and incubate cells at 37C for 1 h and then spread
cells onto LB plates containing appropriate antibiotic. Select
individual clones, inoculate into 100 mL of LB media, and
incubate overnight at 37C. - For AMPK expression, inoculate 1 L of auto-induction
medium with 10 mL of overnight culture, and incubate at
37 C with shaking at 180 rpm, until absorbance at 600 nm
is0.4. Reduce temperature of incubator to 25C and incu-
bate for a further 18 h (seeNote 7). Larger cultures may also be
used, depending on protein recovery and other requirements.
Recover cells by centrifugation at 7000gfor 30 min at 4C,
and freeze the pellets in liquid nitrogen. - For CaMKK2 expression, inoculate 1 L of LB media with
10 mL of overnight culture, and incubate at 37C, shaking
at 180 rpm, until absorbance at 600 nm is0.5. Remove
culture flask, store at 4C for 10 min, add IPTG to a final
concentration of 1 mmol.L^1 , and then reduce temperature to
18 C and incubate flask for a further 18 h. Larger cultures may
Cell-Free Assays for Regulatory Ligands 75