Journal: Biochemical Journal

Article Title: Identification of S-nitrosated mitochondrial proteins by S -nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation

doi: 10.1042/BJ20100633

Figure Lengend Snippet: Mitochondria were untreated or incubated with MitoSNO, and labelled with Cy5 maleimide (red) following either S-nitrosothiol specific reduction ( A , SNO-DIGE) or non-specific reduction ( B , Redox-DIGE). Cy5-labelled samples were combined separately with a Cy3-labelled sample (green) pooled from all replicate experiments and resolved on the same 2D gel. The superimposed fluorescent scans of representative gels from each triplicate experimental condition are shown. Gels used for identification of proteins of interest by MS were subsequently stained with Coomassie Blue ( C ).

Article Snippet: Specific reduction of S -nitrosothiols in the samples was achieved by addition of 1 mM ascorbate and 10 μM CuSO 4 in Chelex 100-treated PBS buffer in the presence of 40 μM CyDye™ DIGE Fluor Cy™3 saturation dye (Cy3) or CyDye™ DIGE Fluor Cy™5 saturation dye [Cy5 (indodicarbocyanine)] (GE Healthcare).

Techniques: Incubation, Two-Dimensional Gel Electrophoresis, Staining