User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2012/02/14
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Objective
DescriptionStocks:
-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
Data
NotesThe 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:
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] |