2.7 Promotion
of Thr172
Phosphorylation
- AMPK, either immunoprecipitated from mammalian cells or
purified from bacterial cells in kinase assay buffer (seeNote 1). - Purified and active LKB1 complex (seeNote 2).
- SDS sample buffer and equipment to run and analyze Western
blots. - Phosphospecific (anti-pT172) and total AMPK antibodies.
- Other reagents as for in-solution kinase assays as Subheading
2.4.
2.8 Protection
Against Thr172
Dephosphorylation
- AMPK, either immunoprecipitated from mammalian cells or
purified from bacterial cells (seeNote 3). - Assay buffer (as for in-solution assays above).
- 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. - AMP or ADaM site activator: 1 mM AMP or ADAM site
activator dissolved in assay buffer (seeNote 4). - MgCl 2 : 50 mM MgCl 2.
- Protein phosphatase: PP1 catalytic subunit (seeNote 5).
- SDS sample buffer and equipment to run and analyze Western
blots. - 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).
- Weigh out sufficient ATP to give 20 mL of 100 mM solution.
- 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.