- Light organelles of two 15-cm dishes of MEFs for each reac-
tion are resuspended with 50μL of fractionation buffer con-
taining 1μg of recombinant GST-ACC1 as substrate at 4C. - Add ATP (200μM final concentration) and/or appropriate
concentrations of desired drugs, e.g., AMP, and incubate the
mixtures on a thermomixer at 800 rpm for 30 min at 30C.
The reactions are terminated by addition of 2SDS and are
analyzed by immunoblotting (described insteps 7– 11 of Sub-
heading3.1).
3.4 Measurement
of AMP, ADP, and ATP
In this section, we describe a CE-MS method, developed by Zhao
JY et al. and Zhao Y et al. [28, 29], to measure the levels of the
cellular adenylate nucleotides. This method is sensitive and effec-
tively protects ATP and ADP from degradation if cautions are
properly taken.
Extraction of AMP, ADP, and ATP from Cultured Cells- MEFs are cultured to 70–80% confluence in 10-cm dishes.
- Aspirate culture medium quickly and rinse cells with 20 mL of
mannitol solution (seeNote 37). - Soak the whole dish in liquid nitrogen, immediately (seeNote
38 ). - Add 1 mL of methanol containing IS1 (1:200) into the dish.
- Scrape off the cells and transfer the cell-methanol mixture in to
a 5-mL reaction tube. Vortex the mixture for 20 s. - Add 1 mL of chloroform and 400μL of ultrapure water into
the mixture, sequentially, and vortex for 20 s after each
addition. - Centrifuge the mixture at 15,000gfor 15 min at 4C.
Collect 450μL of aqueous phase. - The aqueous phase is then ultrafiltrated through an Ultrafree-
MC-PLHCC centrifugal filter at 10,000gfor 3 h at 4C(see
Note 39). - Freeze-dry the ultrafiltrated product.
- Dissolve the dried sample in 100μL of ultrapure water contain-
ing IS2 (1:200). - Load 20μL of sample into the injection vial for the CE-MS
analysis.
Extraction of AMP, ADP, and ATP from Mouse Liver- Mice are starved overnight as described instep 12of Subhead-
ing 3.1. - Anesthetize mouse (seeNote 40).
Analysis of Lysosomal AMPK Activation 405