User:Khyra A. Neal/Notebook/Chem 571/2014/11/04

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November 4, 2014

Tasklist

1. Prepare 45 mM CaCl2 Stock Solution

  • 0.2497 g CaCl2 in 50 mL H2O
  • Prepare 5-35 mM CaCl2 from 45 mM CaCl2 stock solution

2. Measure ISE of CaCl2 Solutions

Ca2+ ISE of Stock Solutions

Concentration Potential measurement (mV)
5 mM 60.8
15 mM 72.8
25 mM 78.1
35 mM 81.3
45 mM 84.3

3. Ca2+ Calibration Curve

  • The data for 5 μM-50mM CaCl2 was combined with 5 mM-45 mM CaCl2 and a final Calibration Curve was formed

Image:Final_Ca2+_Calibration.jpg


4. Prepare dialysis of 30:1 Colloid vs CaCl2 Using 3500 MWCO Tubing

  • Add 1 mL of 30:1 Colloid to one side of the dialysis chamber
  • Add 1 mL of CaCl2 (5 mM-45 mM) to each chamber --- a different concentration per well opposite to 30:1 colloid
  • Sit on low speed shaker overnight to reach equilibrium


5. Preparation of Enzyme Kinetics on UV-Vis

  • This procedure was performed by Alicia Rasines Mazo
    • The 66 mM potassium phosphate solution prepared on October 14, 2014 was used.
    • As recorded, it had pH of 6.319 and it was refrigerated overnight
    • Prepared 10 mL 0.01 wt% Micrococcus lysodeikticus Cell Suspension (in freezer) using phosphate buffer. 0.0012 g of Micrococcus lysodeikticus were added.
    • Adjust the cell suspension concentration until the Absorbance at λ = 450 nm is between 0.6 & 0.7. The protocol followed is listed by Sigma-Aldrich and can be reached here Enzymatic Assay



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