Homework SourseProcedure Preparation of Whole Cell Lysate Note cells and ce
Procedure: Preparation of Whole Cell Lysate
Note: cells and cell lysate have to be kept on ice at all times to prevent proteolysis!
1. Add 200 l of cell lysis buffer (NP-40 buffer: 150 mM sodium chloride 1.0% NP-40 50 mM Tris pH 8.0)
2. Re-suspend the cells by gentle pipetting.
3. Incubate on ice for 30 min, gently vortexing every 5 min.
4. Spin down in microcentrifuge at top speed for 3 min.
5. Transfer the supernatant into a fresh tube. Incubate on ice.
TASK: Calculate final protein concentration in each of the standard (last column in the standard preparation chart).
2
| Tube # | Standard Volume (microliters) | Source of Standard | dH2O Volume (microliters) | Final Protein Concentration (microliters) |
|---|---|---|---|---|
| 1 | 70 | 2 mg/ml stock | 0 | |
| 2 | 75 | 2 mg/ml stock | 25 | |
| 3 | 70 | 2 mg/ml stock | 70 | |
| 4 | 35 | Tube 2 | 35 | |
| 5 | 70 | Tube 3 | 70 | |
| 6 | 70 | Tube 5 | 70 | |
| 7 | 70 | Tube 6 | 70 | |
| 8 | - | - | 70 |
Solution
Calculation
Volume of Protein Standard (microlitre) x Starting Protein Concentration= Amount of protein (microlitre)
Tube
Standard Volume
(microlitre)
Source of standard
dH2O volume
(microlitre)
Final Protein Concentration (microlitre)
1
70
2 mg/ml stock
0
140
2
75
2 mg/ml stock
25
150
3
70
2 mg/ml stock
70
140
4
35
1 mg/ml stock
35
70
5
70
2 mg/ml stock
70
140
6
70
2 mg/ml stock
70
140
7
70
2 mg/ml stock
70
140
8
-
-
70
-
| Tube | Standard Volume (microlitre) | Source of standard | dH2O volume (microlitre) | Final Protein Concentration (microlitre) |
| 1 | 70 | 2 mg/ml stock | 0 | 140 |
| 2 | 75 | 2 mg/ml stock | 25 | 150 |
| 3 | 70 | 2 mg/ml stock | 70 | 140 |
| 4 | 35 | 1 mg/ml stock | 35 | 70 |
| 5 | 70 | 2 mg/ml stock | 70 | 140 |
| 6 | 70 | 2 mg/ml stock | 70 | 140 |
| 7 | 70 | 2 mg/ml stock | 70 | 140 |
| 8 | - | - | 70 | - |
