3.2 Thiols-containing proteins specific labelling
Next, to demonstrate that FL-DT can be linked to thiols-bearing proteins and release fluorescence, bovine serum albumin (BSA), containing 17 pairs of disulfide bonds and one Cys existed as a free sulfhydryl group, 23, 25, 26 was selected as a model protein for the test and probe FL-ST with one single reaction site was used for comparison. As shown in Figure 2A ,FL-ST releases fluorescence upon the addition of BSA by reacting with the single free Cys, while no change in fluorescence is observed in the case of FL-DT . After the disulfide bonds in BSA were reduced with tris(2-carboxyethyl) phosphine (TCEP), the reduced form of BSA (rBSA) was fluorescently labeled with FL-DTsuccessfully (Figure 2A ). The results suggested that, due to the steric effect of BSA, FL-DT could hardly connect the monoCys between two BSA molecules simultaneously; while the released thiol groups from the disulfide bonds would allow the BSA-labeling ofFL-DT . The fluorescent rBSA-FL-DT adducts were then separated and analyzed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). An obvious fluorescent band corresponding to 66.4 kDa was observed for FL-DT treated rBSA, confirming the fluorescently labeled rBSA with FL-DT (Figure 2B ). Taken together,FL-DT is a suitable labeling agent for protein bearing abundant and spatially adjacent Cys residues.