Polyacrylamide Gel Electrophoresis without a Stacking Gel: Application for Separation of Peptides

✍ Scribed by Sung-Kun Yim; Taeho Ahn; Joon-Sik Kim; Chul-Ho Yun

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 61 KB
• Volume: 305
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2002.5597

✦ Synopsis

PAGE 3 systems capable of separating peptides of M r below 10,000 have been reported. Most of these methods have used separation gels with urea (1-4), high concentrated and crosslinked polyacrylamide (5), or a steep polyacrylamide gradient (6, 7) in order to achieve successful resolution of the peptides. High-molarity buffers were also found to be effective for the separation of proteins and peptides in the nonurea SDS-PAGE system (8). Scha ¨gger and von Jagow (9) published a nongradient tricine-SDS-PAGE system without urea, exhibiting high-resolving power in the range of 1-20 kDa. The system was also demonstrated to be suitable for the electrotransfer of separated peptides onto PVDF membranes followed amino acid analysis or NH 2terminal sequence analysis (10). However, the method necessarily needs three gels (separating, spacer, and stacking gels) to achieve sharp bands and high resolution of polypeptides. Each gel layer is made with different concentrations of acrylamide. The use of three gels is tedious and troublesome since each gel mixture must be freshly prepared.