Premium
Optimization of protein sample preparation for two‐dimensional electrophoresis
Author(s) -
Valente Kristin N.,
Choe Leila H.,
Lenhoff Abraham M.,
Lee Kelvin H.
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100659
Subject(s) - sample (material) , electrophoresis , chromatography , chemistry , sample preparation
Optimized 2 DE sample preparation protocols that maximize total protein recovery are fundamental to improving proteome capture and increasing the utility of 2 DE , which is in part limited by inadequate recovery of proteins with diverse physicochemical properties. Maintaining protein solubility is an important factor for protein recovery, but the multitude of solubility‐enhancing agents and the relatively low‐throughput nature of 2 DE limit the systematic study of sample preparation. In this work, design of experiment ( DOE ) approaches are used to optimize protein recovery by altering the levels of four solubility‐enhancing agents (urea, DTT , CHAPS , and SDS ) in the initial suspension solution. Protein recovery is quantified by a total protein concentration assay, which is demonstrated to be representative of SDS ‐ PAGE and 2 DE recovery. DOE methodologies are presented as relatively high‐throughput procedures for optimizing 2 DE sample preparation parameters for a variety of sample types. Optimal suspension solution compositions are shown to vary across a model protein solution (no urea or DTT ), C hinese hamster ovary ( CHO ) cell lysate (8 M urea, ≥2% CHAPS , ≥32.5 mM DTT ), and E scherichia coli cell lysate (8 M urea, 4% CHAPS , 65 mM DTT ), with optimized conditions increasing 2 DE protein recovery at least 50% compared to suboptimal conditions.