- Aspirate the supernatant, and the sediment is the lysosome
fraction. - The purified lysosome can be used for IB by mixing with ten
volumes of 2SDS sample buffer or IP analysis of the lyso-
somal AMPK-activating complex.
Preparation of DRM
In this section, the method for isolating DRM is described. Of
note, DRM can be isolated from light organelles after in vitro
reconstitution assays (Fig.1). All procedures, unless indicated oth-
erwise, should be carried out at 4C or on ice.- MEFs collected from five 10-cm dishes (60–80% confluence),
or 0.3 g of freshly excised liver tissue (handled as described in
steps 12– 14 of Subheading3.1) is homogenized in 2 mL of
Buffer A. - Incubate the homogenates on rotator at 40 rpm for 1 h in
cold room. - The homogenates are mixed well with 2 mL of 80% sucrose
solution by gentle pipetting (seeNote 29). - Load the mixture to the bottom of a 1489-mm centrifuge
tube and overlay sequentially with 5 mL of 35% and 1 mL of 5%
sucrose solutions. - Install each tube onto precooled SW41 rotor, and then sepa-
rate the mixture by ultracentrifugation at 100,000gfor 16 h. - The gradient is collected into ten fractions, 1 mL each. Frac-
tions 2 and 3 contain DRM, and fractions 7–10 (the bottom
fractions) are cytosolic fractions (non-DRM). - Dissolve DRM proteins with an equal volume of DRM buffer,
and then add the same volume of 2SDS sample buffer for
immunoblotting (described insteps 7– 11 of Subheading3.1;
seeNote 30), or for IP with AXIN or LAMTOR1 antibody.
Table 1
Preparation of OptiPrep density gradient medium solutions
OptiPrep (final concentration, %) OptiPrep (mL) 20dilution buffer (mL) 2.3 M sucrose (mL)
27 4.5 0.245 0.6
22.5 3.75 0.282 0.62
16 2.67 0.334 0.65
12 2 0.364 0.71
8 1.33 0.395 0.77Add ultrapure water to 10 mL final volume. Store at 4C
Analysis of Lysosomal AMPK Activation 401