User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/11/05

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Tasks for November 5

  • Enzymatic assay for lysozyme
  • To carry out dialysis data analysis of solution from 11/4

Enzymatic assay for lysozyme

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
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