AMPK Methods and Protocols

2 Materials

Prepare all solutions using ultrapure water (Milli-Q purified demi-

neralized water) and analytical grade reagents. Prepare and store all

reagents at room temperature (unless indicated otherwise).

2.1 Muscle
Homogenization

1. Homogenization buffer: 50 mM HEPES, pH 7.5, 10% (v/v)
   glycerol, 20 mM Na-pyrophosphate, 150 mM NaCl, 1% (v/v)
   Nonidet P-40 (Igepal CA-630), 20 mM beta-glycerophosphate,
   10 mM NaF, 2 mM phenylmethylsulfonyl fluoride, 1 mM
   EDTA, 1 mM EGTA, 10μg/ml aprotinin, 10μg/ml leupeptin,
   2mMNa 3 VO 4 , 3 mM benzamidine. This buffer is stored at
   4 C for up to 1 day.


2. Homogenizer: Retsch TissueLyser II (Qiagen).


3. Rotation apparatus for Eppendorf tubes. Should be placed at
   4 C.


4. Protein determination: Bicinchoninic acid kit.

2.2 Immuno-
precipitation

1. IP buffer (seeNote 1): 20 mM Tris-base, pH 7.4, 50 mM NaCl,
   1% (v/v) Triton X-100, 50 mM NaF, 5 mM Na-pyrophosphate,
   500 μM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol,
   4 μg/ml leupeptin, 50μg/ml soybean trypsin inhibitor, 6 mM
   benzamidine, 250 mM sucrose. This buffer is stored at 4C.


2. Protein A or G agarose (seeNote 2) washed thrice in IP buffer
   and used as a 50:50 slurry suspension. Stored at 4C.


3. IP wash buffer: 120 mM HEPES, pH 7.0, 240 mM NaCl. This
   buffer is stored at 4C.


4. 6sample buffer (6SB): 320 mM Tris–HCl, pH 6.8, 550 mM
   dithiothreitol, 315 mM SDS, 27% (v/v) glycerol, 100–200μg/
   ml bromophenol blue sodium salt. This buffer is stored at
    20 C. To get a 2sample buffer (2SB), dilute the 6SB
   three times in water.


5. Primary antibodies against the various AMPK subunit isoforms
   (seeNote 3).

3 Methods

3.1 Muscle
Homogenization

1. For studies of rodents, it is often possible to dissect well-
   defined muscles or parts hereof. By visual inspection associated
   adipose or connective tissue can be removed. Similarly, muscles
   can be freed of superficial blood contamination by washing in
   ice-cold phosphate-buffered saline. Human muscle samples
   obtained by needle biopsies do not offer a fast procedure for
   such sample cleaning—except a quick rinse in ice-cold buffer.

206 Jesper B. Birk and Jørgen F. P. Wojtaszewski