second piece of transparency sheet over the gel and then secure
with laboratory tape (Fig.2).- (Optional) Use gel drying equipment to dry the gel. This may
increase the radiation signal but is not always necessary (see
Note 18). - Place the gel with transparency sheet into an autoradiography
cassette. In a dark room, position three to five autoradiography
films over the gel and allow exposure to occur from 1 h to
Fig. 1Analysis of kinase assay loading. Coomassie stained gel containing AMPK and target protein substrates.
MW indicates the lane containing the molecular weight markers. Molecular weights corresponding to each
band are listed to the left of the figure. Protein bands corresponding to purified targets 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 “”
Fig. 2Coomassie gel set up for autoradiography. (a) Side view and (b) top-down view of gel set up for
autoradiography. Individual components are labeled accordingly
Identification and Validation of Novel Targets 105