AMPK Methods and Protocols

substrate, because (for reasons that remain unclear) a higher AMP

dependence is always obtained using the former. With any new

assay format, pilot experiments should be performed to ensure

that the counts obtained are proportional to the amount of kinase

added.

1. Carry out either in-solution kinase assays or IP kinase assays as
   described in Subheadings3.4 or 3.5, but varying the concen-
   tration of AMP or other allosteric activator. The range of con-
   centrations used should be determined by pilot experiments—
   try plotting the results as a semilog plot, i.e., log 10 [activator]
   concentration versus activity. Defining EC 50 as the concentra-
   tion giving half-maximal activation, the range of concentra-
   tions should cover from one to two orders of magnitude
   below to one to two orders of magnitude above the EC 50.


2. Calculate the results as described in Subheading3.4 or 3.5. The
   activities can be expressed either as nmol/min/mg or relative
   to activities obtained in the absence of allosteric activator.


3. At concentrations higher than those that cause activation by
   binding to theγsubunit, AMP begins to inhibit AMPK due to
   competitive binding with ATP at the catalytic site on theα
   subunit [8]. Using suitable software such as GraphPad Prism,
   the data can be fitted to the equation:

Y¼BasalþðÞðÞðÞActivationðÞECBasal 50 þX
BasalX
ðÞðÞActivationðÞIC 50 þBasalX: X

whereYis activity,Basalis the activity in the absence of activa-

tor,Activationis the maximal degree of activation relative to

Basal,Xis the AMP concentration,EC 50 is the concentration

of AMP giving half-maximal activation due to binding to theγ

subunit, andIC 50 is the concentration of AMP giving half-

maximal inactivation due to binding at the catalytic site. If the

activities were expressed relative to those obtained in the

absence of allosteric activator, thenBasalshould be set to a

fixed value of 1. Using activators that do not inhibit at high

concentrations, including most of those binding at the ADaM

site, a simpler equation, which omits the inactivation term, may

be used instead:

Y¼Basalþ

ðÞðÞðÞActivationBasal
BasalX

ðÞEC 50 þX

3.7 Promotion
of Thr172
Phosphorylation

Note that AMPK used for promotion of phosphorylation assays

must be dephosphorylated and inactive (seeNote 13). It is essential

that pilot experiments are performed to ensure that activation in the

assay is proportional to the amount of upstream kinase added. The

user should aim for an increase in basal AMPK activity of three- to

fivefold. In-solution assays are typically performed in 25μL reac-

tions using 0.1μg of bacterially expressed heterotrimer. IP kinase

assays are usually performed in 50μL reactions, with the generation

of starting material essentially as in Subheading3.5.Steps 1– 2 and

80 Fiona A. Fyffe et al.