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Use of the PROTEAN Plus Dodeca Cell for Second-Dimension SDS-PAGE, Rev A

l for running second-dimension SDS-PAGE. However, this system is designed to run only two gels per unit for 2-D electrophoresis. Difficulties occurred when sandwich gels were used in order to increase the number of gels from two to four per run. The particular problems that were associated with the sandwich gel system were vertical smearing or cometing of spots from higher molecular weight proteins, and poor reproducibility of gels. Therefore, I have evaluated the use of the PROTEAN Plus Dodeca cell (Figure 1, catalog #165-4151, 220/240 V; also available in 100/120 V, catalog #165-4150) to run these gels. Using the conditions described, I obtained very high-quality gels that could be used for both image analysis and mass spectrometric identification of proteins.


Methods
Preparation of Isoelectric Focusing Strips
Cells from a human cell line were lysed with 7 M urea, 2 M thiourea, 2% CHAPS, 50 mM Tris, pH 7.5, 0.4% dithioerythritol (DTE). Total cell protein (500 g) was loaded, using sample cups, onto pH 47 IPG strips at 14C. Voltage was applied initially at 200 V and gradually increased to 3,500 V over 20 hr, then maintained at 3,500 V for an additional 4860 hr (Langen et al. 2000).

Gel Casting
Second-dimension gels for the Dodeca cell were cast using the PROTEAN Plus hinged spacer plates, the PROTEAN Plus multi-casting chamber, and the Model 395 gradient former. (The Model 495 gradient former, catalog #165-4121, is now available with a larger volume capacity. This would be a more appropriate choice.) Gradients were formed using Laemmli reagents with 4% glycerol added to the higher percentage acrylamide solution to increase gradi
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