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
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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