Preparation of erythrocytes for GSH/GSSG analysis and SDS-electrophoresis
LYSED
Frozen packed erythrocytes treated with NEM, were lysed with 270 μl deionized water, vortexed and split into three parts for further analysis. For analysis of GSH/GSSG, 200 μl of lysate was mixed with 300 μl of precipitation solution to denature the proteins (5.37 mM EDTA, 5.13 M NaCl, and 0.17 M orthophosphoric acid). After 5 min incubation at room temperature, the samples were centrifuged at 12,000 g for 10 min, and supernatants were collected for further analysis. For gel electrophoresis, 100 μl lysate were diluted further with 100 μl deionized water, and half of this dilution was added to 200 μl 3x non-reducing sample buffer, before diluting it by an additional factor of 50 with 1x sample buffer (6.6 mM Tris, 93 μM bromophenol blue, 675 mM glycerol and 0.5% SDS, pH 6.8). To calculate haemoglobin content, 5 μl of the diluted lysate was added to 1 ml Drabkin's reagent (2 mM KCN, 1 mM K