- Incubate at 37C and spot 10μl (or 5μl if using 25μl) of
reaction mix on labeled p81 papers at 10, 20, and 30 min
(a different paper for each spot). - Wash papers three times in 1% (v/v) orthophosphoric acid
(500 ml per wash). - Pour off acid and rinse with acetone.
- Air-dry samples or use a hair dryer until dry.
- Prepare scintillation vials containing 10 ml H 2 O or scintillation
liquid. - Place paper in appropriate vial and count on^32 P channel for
1 min. - Express results as picomoles of^32 P incorporated per micro-
gram of protein per minute (pmol/μg/min) or as a percentage
of control conditions. The counts should increase in a linear
way over the 30 min period. Also, include a no kinase sample
(just lysis buffer) that should be flat over 30 min and subtract
the counts as background.
Western Blot of (Phosphorylated) AMPK Targets
AMPK is known to phosphorylate serine residues in well-
defined AMPK recognition motifs [27], and it therefore has
multitude of downstream targets (reviewed in [28, 29]). Two
of the best characterized AMPK targets are acetyl-CoA carbox-
ylase (ACC), a metabolic enzyme implicated in fatty acid and
cholesterol synthesis [30], and Raptor (regulatory associated
protein of mTOR complex I), whose inhibition is required for
energy stress-induced cell cycle arrest [31]. Thus, (inhibitory-)
phosphorylation status of ACC (Fig. 2b, reproduced from
Leclerc et al. 2004 with permission fromAPS) and Raptor
has been widely used by us and others to assess AMPK activity
in islets andβ-cells [12, 24, 32]. To determine AMPK activity
through the levels of phosphorylated ACC (pACC) and Raptor
(pRaptor) in islets, Western immunoblot (WB) can be used as
follows: - Handpick 10–100 islets (seeNote 9) into 1.5 ml tubes, keeping
the picked islets on ice. Spin for 2 min at 200 rcf at 4C. Wash
once with ice-cold PBS (add 1 ml of PBS, manually resuspend
the islets and spin for 2 min at 200 rcf at 4C). Remove the
supernatant completely. - Lyse the islets in 50μl of RIPA buffer (containing protease and
phosphatase inhibitors) per sample. Pipette up and down to
facilitate the lysis of the islets and incubate on ice for 20 min.
The lysates can now be frozen for posterior analysis. - Spin the lysate for 15 min at 4C, to pellet DNA and cell debris
(seeNote 16). Add 10μlof6SDS sample buffer to the
supernatant.
Manipulation and Measurement in Islets 423