the kinase and substrate. Other pattern matching algorithms,
including Scansite, utilize position weight matrices to create a
probability score for the identified site. In this method, the strin-
gency for the AMPK target identified depends on the construction
of logical operators within the pattern matching function itself. An
advantage of this strategy is the integration of identified putative
targets into an R script that allows customizable integration with
resources listed on Bioconductor (https://www.bioconductor.
org/) for further categorization. Here, we present a two-step
protocol to identify and to preliminarily validate unrecognized
AMPK targets. The first step utilizes the open-source R software
platform and specific coding required for the mapping of the PCS
to a proteome in search for proteins that contain the AMPK PCS
[6]. The coding includes annotation of peptide sequences with
peptide IDs and known gene symbols that are imported into an
Excel workbook. The second step of the protocol involves prelimi-
nary target validation and describes the use of radiolabeled ATP
([γ-^32 P] ATP) in conjunction with autoradiography to assess the
susceptibility of putative target phosphorylation by AMPK.2 Materials
All reagents should be molecular biology grade prepared with
ultrapure double deionized water with a sensitivity of 18 MΩ-cm
at 25C and should be stored at room temperature unless other-
wise indicated. Proper protective equipment should be worn at all
times including gloves, a lab coat, and an eye protection. Addition-
ally, all preparations using [γ-^32 P]ATP should be performed behind
appropriate radiation shields in an environmental health and safety
(EH&S)-approved area. It is also necessary to follow appropriate
decontamination procedures and dispose all waste materials via
EH&S-approved methods.2.1 Materials for In
Silico Analyses
A Windows-, Mac-, or Linux OS-based computer with a minimum
of 8 gigabytes (gb) of random access memory (ram), 250 gigabytes
of storage, and connected to the Internet.2.2 Materials
for AMPK Kinase
Assay
Kinase Assay- Aluminum foil.
- Purified activated recombinant AMPKαβγ heterotrimer.
Recombinant AMPK complexes can be purified from bacterial,
insect, or mammalian cell lysates (seeChapters1–3, 5, and10)
or purchased commercially [1, 2]. Store at 80 C or accord-
ing to the manufacturer’s instructions, and thaw on ice prior to
use (seeNote 1).
100 Brendan Gongol et al.