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