Protein sample preparation and fluorescence labelling

Protein concentration was determined with 2Dquant kit (GE Healthcare Europe GmbH, Freiburg, Germany). The DIGE fluors –N-hydroxysuccinimidyl ester derivatives of the cyanine dyes, Cy2, Cy3 and Cy5 (GE Healthcare Europe GmbH, Freiburg, Germany) –were prepared using freshly opened N,N-dimethylformamide (Sigma-Aldrich). Protein samples (50 μg) were labelled with 400 pmol of Cy2, Cy3 and Cy5 DIGE fluors in separate tubes on ice for 30 min. An internal standard was created by mixing equal amounts of proteins of all samples and labelling it with Cy2, whereas individual samples were labelled with Cy3 and Cy5. The labelling reaction was quenched by adding 1 μL of 10 mmol/L lysine.