- For immunoprecipitation assays mix the following components
in 1.5 mL microcentrifuge tubes: (1) 20μL of AMPK immu-
noprecipitate; (2) 10μL of 1 mM AMP, ADaM site activator or
assay buffer; (3) 10μL of kinase assay buffer. - Start the reaction at 15 s intervals by adding 10μL of phospha-
tase and incubate in shaking incubator at 30C for 10 min.
Also perform blanks where the phosphatase is omitted. - Terminate the reactions at 15 s intervals by the addition of SDS
sample buffer and run a predetermined quantity of protein on
the gel to obtain good signal by Western blot. - While the above method uses Western blotting as a readout, it is
possible to substitute or supplement this with the AMPK activ-
ity assay, although special consideration should be given to
inhibition of the phosphatase. If kinase assays are to be carried
out, we recommend using an okadaic acid-sensitive phospha-
tase such as PP1γand then using okadaic acid to inhibit the
phosphatase. We have found that washing and dilution may be
insufficient to remove the protein phosphatase (see alsoNote1).
4 Notes
- AMPK for promotion of phosphorylation assays must be
unphosphorylated. Use either naı ̈ve, unphosphorylated, bacte-
rially expressed AMPK or kinase immunoprecipitated from a
cell line that does not express LKB1 (e.g., Hela, G361, or A549
cells). If the AMPK is phosphorylated on Thr172, the user
must first dephosphorylate it, but it is essential that the protein
phosphatase is then completely removed and/or inhibited
prior to the promotion assay. For this reason, we recommend
using an okadaic acid-sensitive protein phosphatase such as
PP1γ[10], rather than PP2Cα. If the AMPK is immunopreci-
pitated, the phosphatase can be removed by extensive washing
of the immunoprecipitate in either case, but to ensure there is
no remaining contamination with phosphatase, when using
PP1γyou can add okadaic acid (25μM final) to inhibit any
residual phosphatase. Okadaic acid completely blocks PP1
action without inhibiting LKB1 or AMPK, but there are no
suitable specific inhibitors of PP2Cα. - We use His-tagged LKB1/STRADα/MO25α complex
expressed in, and purified from, insect (Sf9) cells [25]. - AMPK for protection against dephosphorylation assays must
be phosphorylated at Thr172 and active. The user may either
use bacterially expressed kinase that has been phosphorylated
by an upstream kinase (ensuring that the upstream kinase has
been subsequently completely removed) or kinase
82 Fiona A. Fyffe et al.