Solubilization of delipidated macrophage membrane proteins for analysis by two-dimensional electrophoresis

✍ Scribed by Augusto Simões-Barbosa; Jaime M. Santana; Antonio R. L. Teixeira

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 175 KB
• Volume: 21
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/(sici)1522-2683(20000201)21:3<641::aid-elps641>3.0.co;2-h

✦ Synopsis

Two-dimensional electrophoresis of proteins is often precluded due to the lack of solubilization of cell membrane extracts in an aqueous medium. Various additives and detergents have been used to circumvent the problem, but their efficacy may not be satisfactory. In this study, the removal of lipidic components of the cell membrane extract with chloroform-methanol was used to achieve solubilization. Optimal delipidation was obtained with acetone washings. This procedure increased solubilization of membrane proteins from a murine macrophage cell line, thus showing a substantial improvement in gel resolution. The two-dimensional gels loaded with delipidated extract proved to be free of smearing and horizontal streaking. In addition, other protein spots were revealed that were not detected in the gels loaded with undelipidated cell membrane extract.