Tasks for October 1
- Analysis of Dialysis Solutions from Sept. 30
Analysis of Dialysis Solution
Procedure followed as detailed by Dr. Fox on Oct.1
- 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
- Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
- PS cuvettes, measuring 400 - 800 nm
- Don't forget to run a blank with just Bradford & buffer
- Don't forget to run your undialyzed Lysozyme stock
- Transfer remainder of each cell into 20 mL extraction vials
- Measure Ca2+ using ISE
| Substance
|
mV measurement (mV)
|
| 50mM CaCl2 (4) |
80.8
|
| Lysozyme (4) |
80.3
|
| 500μM CaCl2 (3) |
28.8
|
| Lysozyme (3) |
28.1
|
- Transfer 100 μL to a small volume UV cuvette & measure UV absorption
- Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
- Measure entire 200 - 800 nm range
- Transfer 100 μL to a small volume fluorescence cuvette & measure fluorescence
- Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
- excitation at 280 nm
- Transfer 500 - 700 μL to a 15 mL Falcon tube. Add 2 - 3 times volume of water & measure pH
- You want to add minimum amount of water
- You typically need 1.6 - 2 mL in falcon tube to cover pH probe
- So, if you have only 500 μL left, you'll need a 4x dilution (adding 1.5 mL water)
- Or, if you have 700 μL left, you only need a 3x dilution (adding 1.4 mL water)
Preparation of new dialysis
A new dialysis experiment was prepared using 22,000 MWCO tubing instead of 3500MWCO. <br.>
The wells were matched up in the following way. Note that 1 mL of the following were added to each well:
- 50 mM CaCl2. Opposite to it Colloid 1
- 500 μM CaCl2. Opposite to it Colloid 2
- HPLC water. Opposite to it Lysozyme 3
- 0.25 mM HCl. Opposite to it Lysozyme 4
- 50 mM NaCl. Opposite to it Lysozyme 5
- Inserted screws to prevent evaporation
- Placed on low speed shaker for 1 week
|
Project name
Main project page
Previous entry Next entry
