Biology Reference
In-Depth Information
Chapter 22
Evaluating the Effi cacy of Subcellular Fractionation
of Blast Cells Using Live Cell Labeling and 2D DIGE
Yin Ying Ho , Megan Penno , Michelle Perugini , Ian Lewis ,
and Peter Hoffmann
Abstract
Labeling of exposed cell surface proteins of live cells using CyDye DIGE fl uor minimal dyes is an effi cient
strategy for cell surface proteome profi ling and quantifying differentially expressed proteins in diseases.
Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live
cell labeling followed by visualization of the fl uorescently labeled cell surface proteins and fractionated
proteins within a single 2D gel.
Key words:
Live cell labeling, 2D difference gel electrophoresis, Detergent-based fractionation,
Cell surface proteins, Membrane proteins
1. Introduction
Cell surface proteins play an important role in running cellular
processes and may represent diagnostic and therapeutic targets for
many diseases. Due to the low abundance and highly hydrophobic
nature of membrane proteins, detection and identifi cation is chal-
lenging. Accordingly, highly effi cient methods for the enrichment
of membrane proteins are critical. The major requirement for an
effi cient enrichment strategy is maximal recovery of membrane
proteins with minimal cytosolic contamination of the membrane
fraction. This can be evaluated by live cell labeling (LCL) using
impermeable fl uorescent dyes that selectively label cell surface
exposed proteins. Ideally, the process labels only the cell surface
proteins, which should be enriched in the membrane fraction. LCL
has been tested by Mayrhofer et al. (
1
) to assess the compatibility
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