objective
The objective for today is to begin the synthesis of BSA:L-Cysteine Nanocrystals
Protocol BSA:L-cysteine Nanocrystals
- In test tube mix 180 uL of 5mM Zinc Nitrate and 20 uL of 5mM Manganese Acetate
- Add 200 uL of 40 mM Ammonium Acetate
- Add 200 uL of Protein:ligand in various ratios from 0% to 100% protein
- Adjust pH to 8.2 using roughly 250 uL of 10mM Ammonium Hydroxide
- Incubate at room temp for 1 hour
- Add 200 uL of 5mM Sodium Sulfide drop wise, then cover the test tubes
- A VWR Analog Vortex Mixer was used to vortex each test tube for 30 seconds
- Incubate for five-six days at 37 degrees Celsius
Sample Preparation
- Zinc Nitrate
- 0.0372g in 25mL --> 5mM
- Manganese Acetate
- Stock: 0.034g in 25mL --> 5.55mM
- Final: 9mL stock in 10mL --> 5mM
- Ammonium Acetate
- 0.0321g in 10mL -->41.65mM
- BSA
- .0064 g of BSA in 10mL of water --> 9.69 μM
- L-Cysteine
- .0259 g in 25 mL water --> 8.55 mM
- 11.7 uL in 10 mL water --> 10μM
- Ammonium Hydroxide
- 6.76 μL of 28-30% Ammonium Hydroxide in 10 mL
- Sodium Sulfide
- Stock: 0.0495g in 25 mL --> 8.24mM
- Final: 6.06mL of stock in 10mL --> 5mM
% BSA
|
5 mM Zinc Nitrate (uL)
|
5mM Manganese Acetate (uL)
|
41.65 mM Ammonium Acetate (uL)
|
9.69mM BSA (uL)
|
10mM L-Cysteine (uL)
|
10mM Ammonium Hydroxide (uL)
|
5 mM Sodium Sulfide (uL)
|
Total Volume (mL)
|
0 |
180 |
20 |
200 |
0 |
200 |
1150 |
200 |
1.95
|
10 |
180 |
20 |
200 |
20 |
180 |
1000 |
200 |
1.8
|
20 |
180 |
20 |
200 |
40 |
160 |
1000 |
200 |
1.8
|
30 |
180 |
20 |
200 |
60 |
140 |
1000 |
200 |
1.8
|
40 |
180 |
20 |
200 |
80 |
120 |
1000 |
200 |
1.8
|
50 |
180 |
20 |
200 |
100 |
100 |
1000 |
200 |
1.8
|
60 |
180 |
20 |
200 |
120 |
80 |
1000 |
200 |
1.8
|
70 |
180 |
20 |
200 |
140 |
60 |
1000 |
200 |
1.8
|
80 |
180 |
20 |
200 |
160 |
40 |
1000 |
200 |
1.8
|
90 |
180 |
20 |
200 |
180 |
20 |
1450 |
200 |
2.25
|
100 |
180 |
20 |
200 |
200 |
0 |
1100 |
200 |
1.9
|
Table of BSA:L-Cysteine ratios
mol of protein |
2.00E-09 |
1.80E-09 |
1.60E-09 |
1.40E-09 |
1.20E-09 |
1.00E-09 |
8.00E-10 |
6.00E-10 |
4.00E-10 |
2.00E-10 |
0
|
percentage of protein |
100 |
90 |
80 |
70 |
60 |
50 |
40 |
30 |
20 |
10 |
0
|
percentage of ligand |
0 |
10 |
20 |
30 |
40 |
50 |
60 |
70 |
80 |
90 |
100
|
mol of ligand |
0 |
2.00E-10 |
4.00E-10 |
6.00E-10 |
8.00E-10 |
1.00E-09 |
1.20E-09 |
1.40E-09 |
1.60E-09 |
1.80E-09 |
2.00E-09
|
|