Procedure: Preparation of Whole Cell Lysate Note: cells and cell lysate have to
ID: 313516 • Letter: P
Question
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 02
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 - - 70Explanation / Answer
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
-
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