Analysis of rice proteins using SDS-PAGE shotgun proteomics.
Karlie A Neilson, Iniga S George, Samantha J Emery, Sridevi Muralidharan, Mehdi Mirzaei, Paul A Haynes
Methods in Molecular Biology | Published : 2014
In this chapter we describe the workflow used in our laboratory to analyze rice leaf samples using label-free shotgun proteomics based on SDS-PAGE fractionation of proteins. Rice proteomics has benefitted substantially from successful execution of shotgun proteomics techniques. We describe steps on how to proceed starting from rice protein extraction, SDS-PAGE, in-gel protein digestion with trypsin, nanoLC-MS/MS, and database searching using the GPM. Data from these experiments can be used for spectral counting, where simultaneous quantitation of several thousand proteins can be obtained.