Detection of cystic fibrosis protein by isoelectric focusing and silver staining in serum, dried whole blood, and cultured fibroblasts

Abstract

AbstractExperimental conditions were developed for visualizing cystic fibrosis protein (CFP) in cultured fibroblasts from patients with cystic fibrosis (CF) while improving the visualization of CFP in serum. Serum, whole dried blood and cultured skin fibroblast extracts derived from CF homozygotes, heterozygotes, and normal individuals were submitted to isoelectric focusing (IEF) in polyacrylamide gels using a pH gradient of 7.1 to 9.3 and stained for protein with silver. The results showed an improvement in the separation of bands in the cathodal region over that obtained with a pH gradient of 3.5 to 10 and a better visualization of CFP bands than provided with Coomassie blue. Serum IgG could be removed from the cathodal region by including Protein A on the gel during IEF, thus enabling better visualization of CFP bands. With cultured fibroblast extracts, CFP was only observed when gels were stained with silver. This study presents improved sensitivity of a potential diagnostic tool in CF and its use with cultured skin fibroblasts.