User:Khyra A. Neal/Notebook/Chem 571/2014/10/01

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October 1, 2014 Today's experiment is a continuation of September 30,2014 lab protocol Analysis of Dialysis Solutions Bradford Analysis Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl Dilute to 1 mL by adding 780 μL stock 50 mM Tris/NaCl Measure UV-Vis from 400 nm - 800 nm using polystyrene cuvettes. Run blank of Tris/NaCl buffer Run UV-Vis of undialyzed Lysozyme solution with Bradford reagent Transfer remaining dialysis solutions into 20 mL extraction vials. Measure Ca2+ of dialyzed solutions that contain Ca2+ ion using ISE Lysozyme dialyzed in both 50 mM CaCl2 and 500 μM CaCl2 (a total of 4 solutions) Substance Potential (mV) 50mM CaCl2 (4) 80.8 Lysozyme (4) 80.3 500μM CaCl2 (3) 28.8 Lysozyme (3) 28.1 Fluorescence Dilute each dialyzed lysozyme solution by a factor of 100 Transfer 100 mL of dialyzed Lysozyme solution to small volume fluorescence cuvette Measure fluorescence from 300 nm-550 nm and excitation at 280 nm Clean fluorescence cuvette after each use with 1 N HCl, methanol and HPLC water Dialysis Prepare another dialysis chamber using 20,000 g MWCO tubing On one side add 1 mL of each: HPLC water, 50 mM CaCl2, 500 μM CaCl2, 0.25 mM HCl, 50 mM NaCl On opposite side of well add 1 mL of Lysozyme to the wells directly behind the wells containing HPLC water, 0.25 mM HCl, and 50 mM NaCl Add 1 mL of Lysozyme Colloid to the wells directly behind the wells containing 50 mM CaCl2, 500 μM CaCl2. Insert screws to prevent leaving/evaporation Place on low speed shaker for 1 week