User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2012/02/15

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Contents

Objective

  • Terminate dye reactions from 2/14/2012 by changing pH.
  • Remove excess dye from solutions on by dialysis.

Description

  • Reaction Termination

1. Control Solution (Tube 1)

a. Added 16.8 μL HCl (0.948 M)

b. Placed in shaker for several minutes

c. Checked pH = 3.20

d. Added 300 μL Tris buffer

e. Checked pH = 6.49


2. 166 Au/BSA Ratio Solution (Tube 2)

a. Added 79 μL of HCl (0.948 M)

b. Placed in shaker for several minutes

c. Checked pH = 2.72

d. Added 1 mL Tris buffer

e. Checked pH = 6.50


3. 70 Au/BSA Ratio Solution (Tube 3)

a. Added 32 μL of HCl (0.948 M)

b. Placed in shaker for several minutes

c. Checked pH = 2.92

d. Added 600 μL Tris buffer

e. Checked pH = 6.09


  • Dialysis

Solutions were transferred to SnakeSkin® Dialysis Tubing (Thermo Scientific). Tubing was sealed by clips on both sides. Tubing was then immersed in deionized water in large beaker. Stir bar was used to keep water flowing.

The water for the reaction was replaced six times:

1. 2/16/12 - 9:30 A.M.

2. 2/16/12 - 5:10 P.M.

3. 2/17/12 - 2:15 P.M.

4. 2/18/12 - 1:00 P.M.

5. 2/19/12 - 2:45 P.M.

6. 2/20/12 - 1:15 P.M.

Data

  • Calculations - Volume of HCl needed to terminate dye reaction.

- Tris buffer = 50 mM

- HCl = 0.948 M

  • Control Solution ( Tube 1)

Tris buffer = 321 μL

321 μL * 10^-6 L / 1 μL * .05 M Tris / 1 L =


  • Image:2-15-12-572-1.png


  • Image:2-15-12-572-2.png


  • Image:2-15-12-572-3.png

Notes

The reactions were in the shaker for 24 hours, allowing the dye to tag the lysine residues on the BSA. The pH of the solutions needed to be lowered to terminate the lysine tagging reaction. Tris molecules were neutralized using HCl (equal to the amount of Tris added yesterday). After termination dialysis was performed. The dialysis removes the excess dye which hasn't been able to react with the AuNPs. This purification allows for quantifying labeled residues using fluorescence and UV-Vis spectroscopy.

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