One method of improving the
protein profiling of complex mammalian
proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D)
gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing
protein fractions and diffuse, streaked, and/or trailing
protein spots. In this study, I sought to optimize the solubilization of prefractionated human
cancer cell samples using isoelectric focusing (IEF)
rehydration buffers containing a variety of commercially available
reducing agents,
detergents, chaotropes, and
carrier ampholytes. The solubilized
proteins were resolved on 2-D
gels and compared. Among five tested IEF
rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-
propane sulfonate (
CHAPS) and
dithiothreitol (DTT) provided superior resolution, while that containing
Nonidet P-40 (NP-40) did not significantly affect
protein resolution, and the tributyl
phosphine (
TBP)-containing
buffer yielded consistently poor results. In addition, I found that
buffers containing typically high
urea and
ampholyte levels generated sharper 2-D
gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated
proteins from human
breast cancer tissue MCF-7, across a pH range of 4-6.7.