User:Khyra A. Neal/Notebook/Chem 571/2014/11/05

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November 5, 2014

Tasklist

1. Analyze 30:1 Colloid vs CaCl2 Dialyzed on November 4,2014

  • Bradford Analysis
    • 100 μL Sample
    • 200 μL Bradford Reagent (diluted 1:3)
    • 700 μL 50 mM Tris/ 50 mM NaCl Buffer
    • Run UV-Vis between 400-800 nm

2. UV-Vis and Fluorescence

  • Add 1:100 dilution of sample to clear quartz cuvette
  • Run UV-Vis between 200-800 nm
  • Run fluorometer between 300 nm-550 nm and excitation at 280.

3. ISE Measurements

Ca2+ ISE

Substance Potential measurement (mV)
5 mM CaCl2 (1) 56.9
Colloid 1 59.5
15 mM CaCl2 (2) 68.8
Colloid 2 69.6
25 mM CaCl2 (3) 73.7
Colloid 3 72.5
35 mM CaCl2 (4) 76.0
Colloid 4 76.3
45 mM CaCl2 (5) 77.0
Colloid 5 76.4

4. Enzymatic assay for lysozyme

  • This procedure was performed by Alicia Rasines Mazo
  • Preparation of 400 units/mL lysozyme
  • Prepared 10mL 400 unit/mL of lysozyme
    • 0.0867 g of lysozyme were added to a 10 mL volumetric flask and made up to the mark with cold phosphate buffer.
  • Prepared just before addition to keep solution cold

Enzymatic assay

  • Protocol followed as detailed by Sigma-Aldrich [1], with slight modifications.

Using the kinetic mode in the UV-Vis spectrometer,

  • Autozero, measure a blank containing 2.0 mL of cell suspension, and add 0.1 mL of cold buffer. Record the decrease in A450 for 5 min.
  • Measure a blank containing 2.0 mL of cell suspension, and add 0.1 mL of 400 units/mL lysozyme solution. Record the decrease in A450 for 5 min.
  • Obtain the maximum linear rate (ΔA450/minute) for both the Test and Blank using at least a one minute interval and a minimum of 4 data points.
  • Repeat protocol using 200, 300, and 350 lysozyme units/mL solutions