Journal: The Journal of Pediatrics

Article Title: Birth Prevalence of Homocystinuria in Central Europe: Frequency and Pathogenicity of Mutation c.1105C>T (p.R369C) in the Cystathionine Beta-Synthase Gene

doi: 10.1016/j.jpeds.2008.09.015

Figure Lengend Snippet: Quaternary structure of p.R369C mutant under different expression conditions. The p.R369C mutant was expressed in both E. coli and CHO cell-based systems, crude extracts were electrophoresed in gradient polyacrylamide gels under non-denaturing conditions followed by Western blotting (see Methods for details). A representative gel is shown, the sharply demarcated fractions (CBS) 4 , (CBS) 8 , or (CBS) 12 contain 4, 8, or 12 CBS subunits, respectively; the smear in the high molecular weight fraction contains misfolded CBS. The individual lanes contain either the p.R369C mutant (M) , wild-type CBS (WT) , or blank (B , i.e. either E. coli transformed with empty vector or CHO cells transfected with empty vector pTRE2hyg, respectively). The line “Average amount of tetramers/oligomers” shows the relative amounts of the sum of correctly folded mutant tetramers and oligomers relative to the wild-type CBS enzyme, data are means of 3 independent expressions with SD in parentheses.

Article Snippet: For Western blot analysis, cell lysates containing total protein 10 μg were electrophoresed on 4% to 15% gradient native polyacrylamide gels (BioRad precast gels) with Laemmli buffer system with or without SDS for SDS-PAGE and native-PAGE gel, respectively.

Techniques: Mutagenesis, Expressing, Western Blot, High Molecular Weight, Transformation Assay, Plasmid Preparation, Transfection