AMPK Methods and Protocols

3 – 4 below refer to in-solution and IP kinase assays, respectively.

Step 5is common to both methods.

1. For in-solution assays, mix the following components in
   1.5 mL microcentrifuge tubes: (1) 5μL of AMPK, (2) 5μL
   of 1 mM AMP or assay buffer, (3) 5μL of upstream kinase
   (LKB1 or CaMKK2), (4) 5μL of kinase assay buffer (Subhead-
   ing 2.4).


2. Start the reaction at 15 s intervals by adding 5μL of MgCl 2 /
   ATP (25 mM/1 mM), vortex, and incubate at 30C for
   10 min. Also perform blanks where upstream kinase is omitted.


3. For immunoprecipitation assays, mix the following compo-
   nents in 1.5 mL microcentrifuge tubes: (1) 20μL of AMPK
   immunoprecipitate, (2) 10μL of AMP (1 mM) or kinase assay
   buffer, (3) 10μL of upstream kinase (LKB1 or CaMKK2).


4. Start the reaction at 15 s intervals by adding 10μL of MgCl 2 /
   ATP (25 mM/1 mM) and incubate in shaking incubator at
   30 C for 10 min. Also perform blanks where upstream kinase
   is omitted.


5. 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 quantitative signals for Thr172 phosphoryla-
   tion by Western blotting. Alternatively, Thr172 phosphoryla-
   tion can be monitored by kinase assays (seeNote 14).

3.8 Protection
Against Thr172
Dephosphorylation

AMPK used in dephosphorylation protection assays must be phos-

phorylated on Thr172 and thus active (seeNote 15). Pilot experi-

ments should be performed to ensure that the amount of

phosphatase used in the assay gives around 70–80% dephosphory-

lation/inactivation, so that any inhibition of dephosphorylation

will be evident. In-solution assays are typically performed in

25 μL reactions with 0.1μg of phosphorylated bacterially expressed

protein. Immunoprecipitation assays are usually performed in

50 μL reactions with the generation of starting material essentially

as in Subheading3.5.Steps 1– 2 and 3 – 4 refer to in-solution and

IP kinase assays, respectively.Step 5refers to terminating the

reaction and analyzing the results.

1. For in-solution assays mix the following components in 1.5 mL
   microcentrifuge tubes (if the phosphatase is cation-dependent,
   e.g., PP2Cα,Mg2+must also be included in the dephosphory-
   lation mixture): (1) 5μL of AMPK; (2) 5μL of 1 mM AMP or
   assay buffer; (3) 10μL of kinase assay buffer.


2. Start the reactions at 15 s intervals by adding 5μL of phospha-
   tase, vortex, and incubate at 30C for 10 min. Also perform
   blanks where the phosphatase is omitted.

Cell-Free Assays for Regulatory Ligands 81