Tasks for October 28
- To continue analysis of dialysed 0.6 g/ L Lysozyme vs KI using 3500 MWCO
- To check I- ISE values obtained on OCt. 21 and measure KI content of dialysed solutions
- Set up dialysis of 0.6 g/L lysozyme vs CaCl2 using 3500 MWCO
I- ISE
KI concentration
|
mV measurement (mV) (21 Oct.)
|
mV measurement (mV) (28 Oct.)
|
2mM |
-198.3 |
-192.8
|
5 mM |
-217.3 |
-206.8
|
10 mM |
-237.0 |
-224.2
|
25 mM |
-247.4 |
-253.0
|
50mM |
-278.0 |
-276.2
|
0.6 g/L lysozyme vs CaCl2 using 3500 MWCO dialysis data
Bradford Analysis
- Only running bradford analysis of protein-containing solutions, that is Lys 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO.
- Remove 50 μL of solution from each chamber (7 in all) and run Bradford analysis
- Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
- Recall, 50 μL solution + 200 μL diluted Bradford + 750 μL Tris/NaCl buffer
- PS cuvettes, measuring 400 - 800 nm
Ca2+ ISE
CaCl2 concentration
|
mV measurement (mV)
|
5μM (1) |
11.8
|
Lys (1) |
4.6
|
50μM (2) |
4.2
|
Lys (2) |
0.1
|
500μM (3) |
27.4
|
Lys (3) |
24.4
|
5mM (4) |
52.1
|
Lys (4) |
51.3
|
50mM (5) |
77.3
|
Lys (5) |
77.0
|
UV-Vis Absorption
UV-Vis Absorption <br.>
Transfer 100 μL of 100:1 diluted dialysed lysozume to a small volume UV cuvette & measure UV absorption
- Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
- Measured entire 200 - 800 nm range. (Care about peak at 280 nm. Need to compare to fluorescence data to see how the binding is affected)
Fluorescence
Fluorescence
- Measured fluorescence of dyalised solutions
- Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions
- 10μL of lysozyme were diluted to 1mL by adding HPLC. (Remember to account for dilution factor later on)
|