Evaluating the Efficacy of Subcellular Fractionation of Blast Cells Using Live Cell Labeling and 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 320

Fig. 1. Overview of the labeling workfl ow for the LCL/2D DIGE approach.
of CyDye DIGE fl uor minimal dye labeling of human cell lines
in vitro as well as complex biological system in vivo. The effective-
ness of labeling cell-surface-exposed proteins can be assessed by
confocal microscopy. The consistency, however, of LCL needs to
be assessed due to the fact that the optimal pH for CyDye labeling
is between pH 8 and 9, and incubating human cells in nonphysio-
logical conditions over the labeling period may interfere with cell
viability. Decreased cell viability during LCL will affect the evalua-
tion process as compromised membranes become accessible to
CyDyes resulting in possible labeling of cytosolic proteins.
Here, we demonstrate by means of LCL in association with
2D difference gel electrophoresis (2D DIGE), a method for assess-
ing the quality of a membrane fractionation process that resulted in
a majority of the live cell labeled proteins becoming enriched in the
membrane fraction. The observation was consistent over three
technical replicates. An overview of the experimental procedure is
presented in Fig. 1 . In brief, a depleted population of human cells
was split into three groups, labeled with Cy3 in vitro and then
fractionated using a commercially available kit into membrane and
cytosolic fractions. Following Cy5 labeling of each fraction, labeled
proteins were separated by 2D electrophoresis (2DE). The result-
ing gels were imaged for Cy3 (LCL) and Cy5.
A recommendation for the inclusion of an internal standard in
the experimental design is discussed (see Note 1). The inclusion
of an internal standard could minimize experimental variation by
providing a means to standardize the data between analytical gels
while improving spot matching and quantitation.