Tasks for October 7
- Run Bradford Analysis of dialysed solutions from Oct. 1
- Prepare 0.6g/L lysozyme stock
- Prepare new lysozyme colloid
Bradford Analysis of Dialysed Solutions
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
Note: A new 50 mm NaCl 50 mM Tris buffer solution was made by adding:
- (50×10-3mol/L)×(0.050L)×(121.14g/mol)=0.3030g of Tris buffer
- (50×10-3mol/L)×(0.050L)×(58.44g/mol)=0.1461g of NaCl
- And making up to 50 mL with distilled water
pH measurements
| Substance
|
pH measurement
|
| 0.25 mM HCl (4) |
8.062
|
| Lysozyme (4) |
8.134
|
Ca2+ ISE for solutions containing Ca2+
| Substance
|
Potential (mV)
|
| Colloid 1 |
73.5
|
| 50 mM CaCl2 (1) |
74.9
|
| Colloid 2 |
51.2
|
| 500 μM CaCl2 |
91.4
|
Preparation of new lysozyme colloid
A fresh lysozyme stock solution was prepared, given that 1g/L makes the colloid precipitate out of solution. The fresh stock solution is to have a concentration of 0.6g/L, keeping the [Au]:[Lysozyme]=30:1.
- Transferred 0.5 mL 2.5×10-3M HAuCl4 into 5 culture tubes
- Max. protein concentration (double volume of protein as gold)=250μM/6=42μM
- Since final volume of lysozyme stock solution needs to be 25 mL,
- (42×10-6mol/L)*(0.025L)*(14,307g/mol)=0.015g of lysozyme
- Made up to 50 mL mark with distilled water
- Added 1 mL from 42 μM lysozyme stock solution to make [Au:Lysozyme]=30
- Filled up culture tubes to 5 mL total with water
- Placed in oven at 80ºC for 4h
|
Project name
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