Two-Dimensional PAGE of High-Molecular-Mass Proteins

Abstract

Many high-molecular-mass proteins (MW>100 kDa) are known to be involved in cytoskeleton, defense and immunity, transcription and translation in higher eukaryotic organisms. Though a variety of protein separation techniques have been described, at the moment purification of a high-molecular-mass protein remains a difficult task. O’Farrell (1) was the first to devise a two-dimensional gel electrophoresis (2-DE) technique which could detect more than 1000 spots in a gel. Though it is evident that this method was quite powerful in his days, the method is not particularly suitable in analyzing high-molecular-mass proteins larger than 200 kDa. When skeletal muscle, for example, is simultaneously analyzed by sodium dodecyl sulfite (SDS)-polyacrylamide gel electrophoresis (PAGE) and O’Farrell’s 2-DE method, myosin heavy chain (approx 200 kDa) is clearly seen in the former gel, but not in the latter. Hirabayashi (2) was the first to develop a 2-DE method which could analyze high-molecular-mass proteins, including myosin heavy chain and dystrophin, as large as 500 kDa (3). His trick was the use of agarose gel instead of polyacrylamide gel for the first-dimensional isoelectric focusing (IEF). Agarose gel, when used for IEF, can analyze much larger proteins than the polyacrylamide gel can. Oh-Ishi and Hirabayashi (4) further improved the method by adding 1 M thiourea and 5 M urea in an agarose IEF medium. Thiourea is a potent protein solubilizing reagent especially effective for high-molecular-mass proteins that could enter the first-dimensional agarose IEF gel. Here, we describe the 2-DE method with agarose gels in the first dimension (agarose 2-DE) that is compatible with analyzing high-molecular-mass proteins.