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Analysis of skin fibroblast proteins in Duchenne muscular dystrophy: 2. Isoelectric focusing under dissociating conditions
Author(s) -
Burghes Arthur H. M.,
Dunn Michael J.,
Statham Helen E.,
Dubowitz Victor
Publication year - 1982
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150030403
Subject(s) - urea , isoelectric focusing , chemistry , duchenne muscular dystrophy , acrylamide , chromatography , isoelectric point , polyacrylamide gel electrophoresis , immobilized ph gradient , quantitative analysis (chemistry) , gel electrophoresis , biochemistry , enzyme , biology , organic chemistry , monomer , polymer , genetics
Protein profiles of skin fibroblasts labelled with [ 35 S]‐methionine from patients with Duchenne muscular dystrophy (DMD) have been compared with those of cells from normal individuals by isoelectric focusing on thin polyacrylamide gels containing denaturing and solubilising agents cast on silanized supports. The routine method of sample solubilisation used a mixture of 8 M urea and 2 % NP‐40. The inclusion of sodium dodecyl sulphate (SDS) in the sample was found to result in removal of non‐ionic detergent from the gel even if a competition step had been used. In the presence of SDS less material remained at the point of sample application with a concomitant increase in bands in the middle region of the gels. Gels containing a mixture of sulfobetaine and NP‐40 were found to be inferior to those with 8 M urea and 2 % NP‐40, and high levels of urea were found to precipitate the zwitterionic detergent. No consistent qualitative differences between normal and DMD patterns were observed using any of the solubilisation methods on pH 3 to 10 or pH 8 to 10.5 gels. In order to facilitate quantitative analysis by gel slicing techniques, a plastic sheet was developed which would reliably bind acrylamide in the presence of 8 M urea and 2 % NP‐40. The quantitative analysis revealed significant differences between normal and DMD profiles, but the relevance of these changes to the disease state awaits further investigation.