To isolate high molecular weight (HMW) or low-abundance proteins we exploited the high resolving power provided by the molecular sieves of poly-acrylamide gel matrices. Rice-leaf protein extracts were applied to a single well of an SDS-polyacrylamide gel with prestained molecular size markers at both ends. After electrophoresis, the gel was cut into 4 seg-ments according to size, and each segment was ground in extraction buffer. The eluted proteins were sepa-rated from the gel matrix by centrifugation followed by acetone precipitation, and the precipitated proteins were subjected to SDS-PAGE and 2-DE. The SDS-PAGE-based prefractionation method provided non-overlapping discrete sample pools. About 27% more protein spots were detected in the fractionated samples than in the unfractionated samples, and 17% were enhanced. The improvement was especially prominent in the case of HMW proteins. Well-separated HMW proteins were analyzed by MALDI-TOF mass spec-trometry. The molecular masses of the identified pro-teins in the > 48 kDa gel segment were distributed be-tween 50 and 112 kDa, thus validating this prefrac-tionation method. Identified HMW proteins with simi-lar mass but different pI were mostly isoforms. Thus SDS-PAGE-based size prefractionation provides im-proved separation and detection of HMW proteins.

Keywords: 2-DE, High Molecular Weight, Isoforms, MALDI-TOF, Proteome, SDS-PAGE