AMPK Methods and Protocols

22. Autoradiography film: commercially available.


23. Film fixative and developer: commercially available.


24. Automated film developer (optional) or two plastic trays: must
    be large enough to accommodate the autoradiography film.

3 Methods

3.1 Identification
of Proteins Containing
AMPK Consensus
Sequence

1. Install R statistical software from the comprehensive R archive
   network (CRAN) (https://cran.r-project.org/).


2. Once R is installed, install and open RStudio (https://www.
   rstudio.com/).


3. Operating within RStudio, the following packages should be
   installed by entering the following lines of code at the console
   (seeNotes 5and 6 ):

source("http://bioconductor.org/biocLite.R");

biocLite("Biostrings");

source("https://bioconductor.org/biocLite.R");

biocLite("biomaRt");

install.packages("data.table");

install.packages("stringr")

4. Load required libraries by running the following code at the
   console in RStudio (seeNote 7):

library(Biostrings); library(data.table); library(stringr); library(biomaRt)

5. Download proteomes from ensemble:
   Files downloaded from http://uswest.ensembl.org/info/
   data/ftp/index.html. Other proteome sources can be used; how-
   ever, the files need to be organized in FASTA format. Alternatively,
   the human GRCh38 proteome can be downloaded by running the
   following code at the console in RStudio (seeNote 8):

URL<- "ftp://ftp.ensembl.org/pub/release

87/fasta/homo_sapiens/pep/Homo_sapiens.GRCh38.pep.all.fa.gz";

download.file(URL, destfile = "enter file path here/Human_Proteome.fa.gz")

6. Load downloaded proteomes into R by running the following
   code at the console in RStudio (seeNotes 9and 10 ):

proteomehuman<- readAAStringSet ("Human_Proteome.fa.gz ","fasta")

7. Identify protein sequences containing at least one variation of
   the AMPK consensus sequence by running the following code
   at the console in RStudio [1]:
   myhitshuman<- proteomehuman[grepl("(K|R|H)(L|V|M|I|A|F)....(S|T)...(I|L|V|M|A|F)|
   (L|V|M|I|A|F)(K|R|H)...(S|T)...(I|L|V|M|A|F)|

102 Brendan Gongol et al.