- Synthetic peptide substrate [either SAMS
(HMRSAMSGLHLVKRR [21]) or AMARA (AMARAA-
SAAALARRR[22])], 1 mM in kinase assay buffer. - AMP: 1 mM AMP in kinase assay buffer.
- Radioactive Mg.ATP: [γ-^32 P]ATP 150–300 cpm/pmol, 1 mM
ATP, 25 mM MgCl 2. - Phosphoric acid: 1% (v/v) phosphoric acid.
- Scintillation counter and nonaqueous scintillation fluid.
- AMPK, diluted appropriately in kinase assay buffer.
2.5 Immuno-
precipitate (IP) Kinase
Assay
- Benchtop refrigerated centrifuge.
- Roller mixer.
- Benchtop air incubator suitable to hold small orbital shaker.
- IP buffer: 50 mM Tris–HCl, pH 7.4 at 4C, 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,
5 μg/mL soybean trypsin inhibitor, 1% (v/v) Triton-X100.
Immediately prior to use add 1 mM DTT, 0.1 mM benzami-
dine, 0.1 mM PMSF, 5μg/mL soybean trypsin inhibitor, and
1% (v/v) Triton-X100. - Assay mix “plus peptide”: peptide substrate (440μL, 200μM
final concentration in assay); AMP (440μL, 200 μM final
concentration in assay); MgCl 2 /[γ-^32 P]ATP (440μL, 5 mM/
200 μM final concentration in assay). - Assay mix “minus peptide”: assay buffer (220 μL); AMP
(220 μL, 200 μM final concentration in assay); MgCl 2 /
[γ-^32 P]ATP (220μL, 5 mM/200μM final concentration in
assay). - Small orbital shaker (set at about 1000 rpm).
- Protein G-Sepharose.
- Anti-AMPK antibody (any immunoprecipitating antibody
against target subunit or complex can be used; our standard
protocol uses an equal mixture of anti-α1 and anti-α2 antibo-
dies to precipitate total AMPK from cell lysates). - Other reagents as for in-solution kinase assays as in Subheading
2.4. - Cell or tissue lysates containing AMPK.
2.6 Allosteric
Activation by Ligands
Binding the Nucleotide
or ADaM Sites
- Reagents as Subheadings2.4 or 2.5.
Cell-Free Assays for Regulatory Ligands 73