AMPK Methods and Protocols

2.7 Promotion
of Thr172
Phosphorylation

1. AMPK, either immunoprecipitated from mammalian cells or
   purified from bacterial cells in kinase assay buffer (seeNote 1).


2. Purified and active LKB1 complex (seeNote 2).


3. SDS sample buffer and equipment to run and analyze Western
   blots.


4. Phosphospecific (anti-pT172) and total AMPK antibodies.


5. Other reagents as for in-solution kinase assays as Subheading
   2.4.

2.8 Protection
Against Thr172
Dephosphorylation

1. AMPK, either immunoprecipitated from mammalian cells or
   purified from bacterial cells (seeNote 3).


2. Assay buffer (as for in-solution assays above).


3. IP buffer containing phosphatase inhibitors: 50 mM Tris–HCl,
   pH 7.4 at 4 C, 150 mM NaCl, 50 mM NaF, 5 mM Na
   pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,
   0.1 mM benzamidine, 0.1 mM PMSF, 5μg/mL soybean
   trypsin inhibitor, 1% v/v Triton-X100. Immediately prior to
   use add 1 mM DTT, 0.1 mM benzamidine, 0.1 mM PMSF,
   5 μg/mL soybean trypsin inhibitor, 1% v/v Triton-X100.


4. AMP or ADaM site activator: 1 mM AMP or ADAM site
   activator dissolved in assay buffer (seeNote 4).


5. MgCl 2 : 50 mM MgCl 2.


6. Protein phosphatase: PP1 catalytic subunit (seeNote 5).


7. SDS sample buffer and equipment to run and analyze Western
   blots.


8. Phospho-Thr172 and total AMPK antibodies.

3 Methods

3.1 Kinase Assays:
Making Up ATP

To accurately determine the degree of allosteric activation by AMP,

it is essential that the unlabeled ATP used as substrate is free from

contaminating AMP. We recommend preparing a stock solution of

unlabeled ATP at a concentration of approximately 100 mM (which

is stable at
 80 C in frozen aliquots for 1–2 years). The purity of

this stock preparation can be monitored using liquid chromatogra-

phy or capillary electrophoresis (seeChapters15 and 16).

1. Weigh out sufficient ATP to give 20 mL of 100 mM solution.


2. Place 17 mL of kinase assay buffer in a small beaker, and place
   this in a large beaker partially filled with ice (this keeps the
   buffer chilled). Set this stirring, with a clean pH electrode in the
   buffer.

74 Fiona A. Fyffe et al.