User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2012/02/14

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Contents

Objective

  • Begin protein labeling reaction using 5(6) - Carboxynaphthofluorescein, N-succinimidyl Ester dye to tag lysine residues in Au/BSA reaction mixtures.

Description

Stocks:

  • Au/BSA mixtures were synthesized on 2/1/2012
  • 50 mM Tris buffer solution was synthesized on 2/8/2012
  • 17.7 μM control solution of BSA was synthesized on 2/8/2012


Reaction:

  • 1. Adjusting pH of solutions to 8 - 9 range

-BSA Control

Initial: pH = 3.98, Volume = 10 mL

Added 321 μL of Tris Buffer

Final: pH = 8.50, Volume = 10.32 mL

-Purple Fiber Solution (Au/BSA = 166)

Initial: pH = 3.17, Volume = 10 mL

Added 1.5 mL Tris Buffer

Final: pH = 8.51, Volume = 11.5 mL

-Purple Solution (Au/BSA = 70)

Initial: pH = 3.50, Volume = 10 mL

Added 600 μL Tris Buffer

Final: pH = 8.50 , Volume = 10.6 mL

  • 2. Three dye solutions were prepared using 2 mg of dye in 160 μL of DMSO
  • 3. One dye solution was added to the BSA control, another to the purple solution (Au/BSA = 70), and the last one to the purple fiber solution (Au/BSA = 166)
  • 4. All three solutions were placed in shaker at room temperature for 24 hours.

Data

  • As soon as the dye solutions were added to the mixtures and control, an immediate color change was observed.
  • Tube 1 was the BSA control. Initially it was a clear solution, and after the dye was added a gradient was observed that went from purple near the bottom of the tube to blue at the surface.
  • Tube 2 was the purple fibers in clear solution. After adding the dye, the entire solution turned blue
  • Tube 3 was the purple solution. After adding the dye, the solution formed a color gradient from red near the bottom of the tube, to purple at the surface.


Image:2-14-2012-1.jpg

Notes

The dye being used is 5(6)-Carboxynaphthofluorescein, N-succinimidyl ester which has been obtained from Marker Gene Technologies Inc. This dye is capable of tagging unprotonated lysine residues. In order to deprotonate the lysine we needed to increase the pH of our solutions using Tris Buffer (control, 70 ratio, 166 ratio) to reach pH 8.5 (because the lysine pKa = 8.95 for the amino group). The dye is insoluble in water, therefore it was first dissolved in as little DMSO as possible and then added to the Au/BSA mixtures. The reaction will be left overnight; with dialysis beginning tomorrow to remove the excess dye from the mixtures.

Dye Reaction Mechanism:

Image:2-14-2012-2.jpg


  • References:

Steven J. Bark and Klaus M. Hahn, Fluorescent Indicators of Peptide Cleavage in the Trafficking Compartments of Living Cells: Peptides Site-Specifically Labeled with Two Dyes, METHODS 20, 429–435 (2000)

"Dye labelling protocol": [1]

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