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Today's objective is to perform a dialysis using the solution we made yesterday. The reaction has been running for 24hours in the shaker at room temperature, allowing the dye to tag our BSA peptides by binding unprotonated lysine. The dialysis is going to allow us to remove the excess of dye which hasn't been able to react with our Gold NPs (because number of available Lys site is limited). This step allows us to purify our solution so that we'll then be able to quantify labelled NPs using UV-Vis spectrum; and the efficiency of the dying process will be determined using fluorescence spectroscopy.
The first step of today's experiment is to lower the pH of our solution so that the reaction stops; to do so, we have to neutralize Tris molecules using HCl (we have to add an equal amount of HCl to the amount of Tris we added yesterday). Then we'll perform a 7 days dialysis to purify our solution. We are going to save pH values at every step of the protocol because we believe this parameter is crucial.
The dialysis was performed using SnakeSkin® Dialysis Tubing (Thermo Scientific)
Calculations for the volume of HCl needed to neutralize Tris.
Concentration of Tris buffer = 50mM
Concentration of HCl solution = 0.948M
Volume of Tris buffer = 321µL
=> Number of mole of Tris = 321 x 10^-6 x 50 x 10^-3 = 16µM
=> Volume of HCl solution to add = (1.6 x 10^-5)/0.948 = 16.8µL
Volume of Tris buffer = 1.5mL
=> Number of mole of Tris = 1.5 x 10^-3 x 50 x 10^-3 = 75µM
=> Volume of HCl solution to add = (7.5 x 10^-5)/0.948 = 79µL
Volume of Tris buffer = 600µL
=> Number of mole of Tris = 600 x 10^-6 x 50 x 10^-3 = 32µM
=> Volume of HCl solution to add = (3.2 x 10^-5)/0.948 = 32µL