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.