Two-dimensional gel electrophoresis of actin-binding proteins isolated by affinity chromatography from human skeletal muscle

✍ Scribed by Dr. Joëlle V. F. Coumans; Ian Humphery-Smith; Cristobal G. dos Remedios

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 697 KB
• Volume: 18
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150180709

✦ Synopsis

In muscle cells actin exists as a mixture of monomeric (G-actin) and filamentous actin (F-actin) and ionic conditions strongly favor the formation of F-actin. The existence of unpolymerized actin depends, among other factors, on proteins that bind to G-actin, the so-called G-actin-binding proteins (G-ABPs). We have coupled monomeric actin to divinylsulphone-activated agarose (Mini-Leak) to isolate G-ABPs in human skeletal muscle. Eluted proteins were analyzed by two-dimensional gel electrophoresis (2-DE), which shows that some proteins are selectively retained. Deoxyribonuclease I (DNase I) is known to bind residues at the "pointed end" of actin (subdomains 2 and 4) with a high affinity. When DNase I is bound to the actin Mini-Leak before applying the skeletal muscle extract, the 2-DE gels of the eluted proteins reveals differences when compared to gels of proteins eluted from actin-Mini-Leak and DNase I-Mini-Leak affinity columns. This strategy should detect ABPs which bind to sites other than the DNase I-binding site and some may prove to be novel.