October 1, 2014
Today's experiment is a continuation of September 30,2014 lab protocol
Analysis of Dialysis Solutions
Bradford Analysis
- Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis
- Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
- Dilute to 1 mL by adding 780 μL stock 50 mM Tris/NaCl
- Measure UV-Vis from 400 nm - 800 nm using polystyrene cuvettes.
- Run blank of Tris/NaCl buffer
- Run UV-Vis of undialyzed Lysozyme solution with Bradford reagent
- Transfer remaining dialysis solutions into 20 mL extraction vials.
- Measure Ca2+ of dialyzed solutions that contain Ca2+ ion using ISE
- Lysozyme dialyzed in both 50 mM CaCl2 and 500 μM CaCl2 (a total of 4 solutions)
| Substance
|
Potential (mV)
|
| 50mM CaCl2 (4) |
80.8
|
| Lysozyme (4) |
80.3
|
| 500μM CaCl2 (3) |
28.8
|
| Lysozyme (3) |
28.1
|
Fluorescence
- Dilute each dialyzed lysozyme solution by a factor of 100
- Transfer 100 mL of dialyzed Lysozyme solution to small volume fluorescence cuvette
- Measure fluorescence from 300 nm-550 nm and excitation at 280 nm
- Clean fluorescence cuvette after each use with 1 N HCl, methanol and HPLC water
Dialysis
- Prepare another dialysis chamber using 20,000 g MWCO tubing
- On one side add 1 mL of each:
- HPLC water, 50 mM CaCl2, 500 μM CaCl2, 0.25 mM HCl, 50 mM NaCl
- On opposite side of well add 1 mL of Lysozyme to the wells directly behind the wells containing HPLC water, 0.25 mM HCl, and 50 mM NaCl
- Add 1 mL of Lysozyme Colloid to the wells directly behind the wells containing 50 mM CaCl2, 500 μM CaCl2.
- Insert screws to prevent leaving/evaporation
- Place on low speed shaker for 1 week
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Project name
