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
|
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