Analytical Sciences

A New Isoelectric Focusing System for Fast Two-Dimensional Gel Electrophoresis Using a Low-Concentration Polyacrylamide Gel Supported by a Loose Multifilament String

Jinxiang LI,* Ayaka OGASAWARA,** Tamao ODAKE,** Tomonari UMEMURA,** and Kin-ichi TSUNODA**

*Department of Chemistry, Liaoning Normal University, 850 Huanghe Road, Dalian 116029, China
**Department of Chemistry, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan

A new isoelectric focusing (IEF) system for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been proposed. In this system, a super-soft and tough IEF gel was achieved by casting polyacrylamide gel down to 2.0% T using a loose multifilament string (LMS) of nylon as a gel support. The IEF apparatus for the LMS-gel, fabricated from acrylic boards, had a cooling water chamber, and eliminated the need of electrode solutions by directly connecting the two ends of individual gels to platinum electrodes. The carrier ampholyte-generated pH gradients using the new IEF system was stable over a long duration of time and a wide range of voltages, and the IEF time became shorter using a 2.0% T gel than using a 4.0% T gel. Also, the LMS-gels prepared in different runs exhibited excellent reproducibility. The new IEF system was applied to 2-D PAGE of a chicken skeletal muscle extract, and it was found that the protein loading capacity, protein entry into the LMS-gels, and protein transfer efficiency from the first-dimensional to the second-dimensional gels were significantly improved by using a low-concentration (2.5% T) gel. Also, proteins of high molecular weight of more than 200 kDa were observed in the 2-D maps, and therefore the new IEF system has a very good potential to be applied for fast 2-D PAGE of high molecular-weight proteins.

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