AMPK Methods and Protocols

In this chapter, we will discuss how cell-free assays can be used

to test the effects of ligands and drugs on the three mechanisms:

(1) allosteric activation, (2) promotion of Thr172 phosphorylation,

and (3) inhibition of Thr172 dephosphorylation, by which AMPK

is regulated.

In the description below, we describe kinase assays using

[γ-^32 P]ATP, which our laboratory prefers due to their great sensi-

tivity, flexibility, and reproducibility. However, the protocols can be

adapted for nonradioactive kinase assays.

2 Materials

Prepare all solutions with ultrapure water. Reagents are stored at

room temperature unless indicated otherwise.

2.1 Kinase Assays:
Making Up ATP
and Determining
Specific Radioactivity

1. Unlabeled ATP: 100 mM ATP.


2. [γ-^32 P]ATP: Around 6000 Ci/mmol.


3. Kinase assay buffer: 50 mM Na HEPES, pH 7.4, 150 mM
   NaCl, 1 mM DTT, 0.02% (v/v) Brij-35. Store at room tem-
   perature, but do not add Brij-35 and DTT until immediately
   prior to use; otherwise, store in aliquots at
   20 C.


4. NaOH (1 mol.L
   ^1 ).


5. pH electrode and meter.


6. UV/visible spectrophotometer.

2.2 Expression
and Purification
of AMPK from
Bacterial Cells

1. Polycistronic bacterial plasmid expressing all three subunits of
   AMPK (e.g., [19]).


2. BL21-CodonPlus (DE3)-RIL bacterial cells.


3. SOC medium: Dissolve to 1 L deionized water, 20 g of tryp-
   tone, 5 g of yeast extract, 2 mL of 5 M NaCl, 2.5 mL of 1 M
   KCl, 10 mL of 1 M MgCl 2 , and 10 mL of 1 M MgSO 4.
   Autoclave at 121C for 15 min and add 20 mL filter sterilized
   1 M glucose.


4. Lysogeny Broth (LB) medium with appropriate antibiotic:
   Dissolve to 1 L deionized water, 10 g tryptone, 10 g NaCl,
   5 g yeast extract, and autoclave at 121C for 15 min. Add
   antibiotic when cooled.


5. LB plates with appropriate antibiotic: LB as above with the
   addition of 12 g of Select agar. When cooled but still fluid,
   add antibiotic and pour into plates.


6. Auto-induction medium with appropriate antibiotic: Dissolve
   10 g tryptone and 5 g yeast extract in 929 mL deionized water
   and autoclave at 121C for 15 min. Once cooled, add appro-
   priate antibiotic, 1 mL of 1 M MgSO 4 ,20mLof50 5052

Cell-Free Assays for Regulatory Ligands 71