Global protein expression profiling of various mutants or growth conditions is currently a major challenge in biology. Here we provide a protocol for a strategy that we recently developed that couples ORFeome-based (ORF denotes open reading frame) expression to reverse protein arrays; this approach accurately quantifies more than 99% of the predicted fission yeast proteins in various genetic backgrounds. The first stage of this two-stage protocol requires mass mating between any fertile fission yeast mutant of interest and the integrated fission yeast-tagged ORFeome followed by selection of recombinant haploids. The second stage of the protocol, called reverse protein arrays, involves simple large-scale extraction of total proteins, which are then spotted on nitrocellulose membranes for detection by quantitative dot blot. When handled manually, the entire protocol takes about 2 months. However, the process could easily be automated and should also be applicable to other organisms.
|Number of pages||6|
|Publication status||Published - 2012|