Tasks for November 5
- To carry out dialysis of 30:1 Colloid solution vs. CaCl2 using MWCO 3500 between 10-50 mM.
- To prepare solutions for enzyme kinetics
Preparation of solutions for enzyme kinetics
- The 66 mM potassium phosphate solution prepared on Oct. 14 was used. As recorded, it had pH of 6.319.
- 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: [1]
Preparation of CaCl2 solutions for dialysis
- A stock solution of 45 mM CaCl2 was prepared by adding 0.2502 g CaCl2 to a 50 mL volumetric flask amd making up to the mark with HPLC water.
- Serial dilutions were made for 35, 25, 15 and 5 mM CaCl2 mM.
Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysis
A new dialysis experiment was prepared using 3,500 MWCO tubing. <br.>
The wells were matched up in the following way. Note that 1 mL of the following were added to each well:
- 5 mM CaCl2. Opposite to it 30:1 Colloid
- 15 mM CaCl2. Opposite to it 30:1 Colloid
- 25 mMM CaCl2. Opposite to it 30:1 Colloid
- 35 mM CaCl2. Opposite to it 30:1 Colloid
- 45 mM CaCl2. Opposite to it 30:1 Colloid
- Inserted screws to prevent evaporation
- Placed on low speed shaker for 1 day
Dialysis Data
- Inspection of the dialysis wells showed that precipitation started to occur at 25 mM of CaCl2, and it was fully precipitated by 35, and 45 mM of CaCl2.
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
|
|
Project name
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