FIG. 5.

Total SNO-protein profiles labeled with biotin-HPDP, using the labeling conditions of the original BST protocol and CyDye switch in the presence and absence of ascorbate. Equal amounts of total placental protein extracts were subjected to biotin switch for labeling total SNO-proteins under different conditions. Lane 1: samples were treated with 1 mM DDT and then subjected to biotin-HPDP labeling. Only 1 μg of total protein (1/20 of the other lanes) was loaded. Lane 2: original BST, without ascorbate removal before labeling; Lane 3: original BST, with ascorbate removal before labeling; Lane 4: BST using CyDye switch condition without ascorbate removal before labeling; Lane 5: BST using CyDye Switch condition with ascorbate removal before labeling. The proteins were resolved with 10% SDS-PAGE, transferred onto PVDF membranes, and then immunoblotted with an anti-biotin antibody.