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This is a fluorescent DIGE analysis of cellular proteins.
The 2-D fluorescent DIGE consisted of normal SDS-PAGE as the first dimension and
Zn2+–Phos-tag SDS-PAGE as the second dimension.
We prepared a set of lysates from untreated HeLa cells and another set from the same cells treated with calyculin A (100nM, 30min), which acts as an inhibitor of serine/threonine phosphatase. Furthermore, the complete
cell lysates were divided into soluble and insoluble protein fractions to reduce the complexity of the migration spots detected on the 2-D gel. In this study, we obtained comparative data from the DIGE analysis by using the insoluble protein fraction. A mixture of HeLa lysate labeled with Cy3 and the calyculin A-treated cell lysate labeled with Cy5 was subjected to the first-dimensional
SDS-PAGE using a 300-mm-long slab gel. After electrophoresis, the first-dimensional gel was cut into three parts corresponding to molecular-mass ranges of 10–30 kDa, 30–50 kDa,
and 50–200 kDa, respectively. Each 1-D gel slice was subjected to a second-dimensional Zn2+–Phos-tag SDS-PAGE on 5.5–9.5% w/v polyacrylamide gels (with the choice of gel depending on the molecular-mass range), containing 50 µM Zn2+–Phos-tag. The images of the gels obtained by detection of Cy3 and Cy5 are represented by magenta and green,respectively; when these two images were superimposed, overlapping spots appeared white in the resulting image.
In the 2-D Phos-tag gel, many magenta and green spots were observed. This observation could be the result of shifts in mobility induced by differences in phosphorylation status, which depended on whether the cells were treated with calyculin A. We estimated that some magenta and/or green
spots aligned in the vertical direction represent multiple phosphorylated species derived from a single protein. We selected six typical spots (#1–#6) and, after silver staining of the same 2-D Phos-tag gel, we performed MS analysis to identify these proteins. As a result of database searches, the proteins corresponding to the spots were identified as
follows:
1#: histone H3 (15 kDa),
2#: keratin 18 (47 kDa),
3#:keratin 8 (53 kDa),
4#: vimentin (54 kDa),
5#: lamin A/C(74/65 kDa),
6#: elongation factor 2 (95 kDa) .