Figure 2

Biotin switch method to detect protein S-nitrosylation. As shown in the flowchart, the biotin switch was performed by a three-step reaction. (A) The biotinylated lysates were subjected to DTT-free (non-reduced) 2-DE that was stained by VisPRO dye to determine translational levels (total protein). Western blotting was subsequently performed by hybridization with streptavidin-HRP to determine posttranslational S-nitrosylation levels. (B) Another separated 2-DE gel was prepared for the pecking of the S-nitrosoproteins and then it was analyzed by mass spectrometry. (C) The biotinylated lysates were also applied to a pull-down assay by neutravidin agarose to assure that the identified proteins were biotinylated (i.e. S-nitrosylated).