AMPK Methods and Protocols

3.2 Analysis
of the Lysosomal
Localization of AXIN/
LKB1

Preparation of Lysosomes

In this section, the method preparation of lysosome is

described. The method was originally developed by Sigma-Aldrich

LLC, with some modifications. All procedures, unless otherwise

indicated, should be carried out at 4C or on ice.

1. Collect MEFs from 60 10-cm dishes (60–80% confluence) by
   directly scraping, at RT (seeNote 26).


2. Centrifuge the cells at 500gat 37C(seeNote 26) for
   5 min.


3. Resuspend the cells in 7 mL of 1Extraction Buffer (diluted
   with ultrapure water) containing protease inhibitor cocktail at
   RT (seeNote 26).


4. Move the suspended cells to ice bucket and immediately break
   the cells in a 7-mL Dounce homogenizer for 120 strokes.


5. Centrifuge the sample at 1000gfor 10 min, yielding post-
   nuclear supernatants (PNS).


6. Transfer the PNS equally to six new tubes. Centrifuge the PNS
   at 20,000gfor 20 min.


7. Suspend the pellet in each tube in 1Extraction Buffer to
   400 μL final volume (seeNote 27) by gentle pipetting. The
   resuspension is the crude lysosomal fraction (CLF).


8. Add 253μL of OptiPrep and 137μLof1OptiPrep Dilution
   Buffer (diluted with ultrapure water) to each CLF in the tube.
   Mix the CLF well by gentle pipetting. The mixture is defined as
   the Diluted OptiPrep Fraction (DOF).


9. Prepare 10 mL of each OptiPrep density gradient medium
   solutions (Table1).


10. Prepare six 1160-mm centrifuge tubes, each sequentially
    loaded with 0.4 mL of 27% and 0.5 mL of 22.5% OptiPrep
    solution, and overlay with 0.8 mL of DOF, and then with 1 mL
    of 16%, 0.9 mL of 12% and 0.3 mL of 8% OptiPrep solution.


11. Install each 11  60-mm centrifuge tube onto precooled
    SW60 Ti rotor, and then separate the mixture by ultracentrifu-
    gation at 150,000gfor 4 h.


12. The fraction at the top of 12% OptiPrep solution (approxi-
    mately 200μL per tubeseeNote 28) is collected as the lyso-
    some fraction.


13. Add CaCl 2 to the lysosome fraction to an 8 mM final concen-
    tration, and centrifuge the fraction at 5000gfor 15 min.
    Move the supernatant to a new tube.


14. Mix the supernatant with two volumes of PBS. Centrifuge the
    mixture at 20,000gfor 20 min.

400 Chen-Song Zhang et al.