the complete DMEM medium directly or administered into
animals at chosen doses.- It should be noted that AMPK could be artificially activated
during collection of cells or dissection of animal tissues. There-
fore, cautions must be taken to avoid elevation of basal AMPK
activities. - According to our experience, never let the cells reach 100%
confluence; the basal activity of AMPK will increase otherwise.
In addition, contamination of mycoplasma would also inflict an
increase of basal AMPK activity. - Rinsing cells with PBS (especially at RT) is not recommended;
it will raise the basal level of activated AMPK, otherwise. - Cells should be kept at ambient temperature before lysis. Cold
shock will directly activate AMPK. - Pay special attention to temperature rising during the sonica-
tion. Hence, sonication should be intermittent. - Decapitation must be avoided to prevent draining of blood,
because AMPK will otherwise be phosphorylated by CaMKKβ
in response to ischemia [31, 32]. - Liver tissue can also be harvested after anesthesia is applied to
mouse. - As an alternative choice, homogenize the frozen tissue in
ice-cold buffer directly. - The cells should be kept at ambient temperature until they are
broken by Dounce homogenizer to avoid AMPK activation by
cold shock. - We find that it is a critical step for lysosome isolation. Make sure
that the pellet is sufficiently suspended and volume of the
suspension should be precisely adjusted to 400μL. - Avoid collecting too much lysosome fraction (e.g., more than
300 μL); it would otherwise reduce the purification efficiency
ofstep 12. - Avoid bubbles when mixing the sample—it is critical for the
isolation of DRM. - If the concentration of protein is too low to be detected,
precipitate the protein by adding trichloroacetic acid to the
DRM fraction (10% (v/v) final concentration). Wash the pre-
cipitant with ice-cold acetone for two times and dissolve the
precipitant with ODG buffer. The sample is then sonicated,
centrifuged, and mixed with SDS sample buffer for immuno-
blotting (described insteps 7– 11 of Subheading3.1). - Dilute the formaldehyde prior to use, and do not rinse the cells
with PBS before fixation. As an alternative choice, add 2 mL of
408 Chen-Song Zhang et al.