2 weeks depending on the radiant intensity of the sample (see
Note 19).- Following exposure, the films can be developed in a dark room
one at a time by running through an automated developer. If
an automated developer is unavailable, develop one film at a
time by submerging in a tray containing developer with gentile
agitation for 1–5 min prior to placing it in the fixative solution
with gentle agitation for 1–5 min. Wash with water and hang to
dry. Positive results will appear as a third band on the autora-
diograph (Fig.3).
4 Notes
- Due to the sensitivity of recombinant proteins to degradation
by freezing and thawing, it is best to aliquot recombinant
proteins into single-use aliquots to minimize the loss of
AMPK activity. - Because [γ-^32 P] ATP has a half-life of 2 weeks, it should be used
soon after arrival for optimal results. - It is recommended to prepare fresh HEPES buffer prior to use,
and the pH should be adjusted to 7.4. - Buffer A, B, and C should be kept on ice.
- For each step, the codes should be entered as single lines at the
console in RStudio. - Refer to Entrosolve.com for programming assistance if
necessary.
Fig. 3Final autoradiography results. Autoradiograph of AMPK alone (left panel) or in the presence of target
substrates (right panel). Autoradiograph bands resulting fromγ-^32 P incorporation into target proteins are
indicated with arrows. Lanes containing AMPK in the reaction are indicated with a “þ” at the bottom of the
gel, while those without AMPK are indicated by a “”. These data indicate that AMPK can phosphorylate the
target substrates in vitro and that phosphorylation of these targets is dependent on AMPK since the target
substrate incubated without AMPK does not illustrate an autoradiograph band
106 Brendan Gongol et al.