Protein Lysates (from cultured cells – protocol from Sylvain)
Solutions:
 1x DPBS (Cellgro, 21-031-CV)
 NP40 Buffer (for final volume of 50 ml):
Reagent:
ddH2O
NaCl 5M
Tris-HCl pH 8.0
glycerol
NP-40
Sigma, 74385
EDTA 0.5M
protease inhibitors
Roche, #11 836 170 001
for 50 ml
25 ml
1370 l
1000 l
5 ml
500 l
for 10 ml
5 ml
274 l
200 l
1 ml
100 l
[Final]
200 l
5 tablets
40 l
1 tablet
2 mM
1 tab/10 ml
phosphatase inhibitors
Roche, #04 906 845 001
5 tablets
1 tablet
1 tab/10 ml
Bring up to 50 ml with
ddH2O
Bring up to 10 ml with
ddH2O
137 mM
20 mM
10%
1%
Procedure:
1. Wash cells with 2 ml cold 1X DPBS, 2-3 times. Keep plates on ice as much as possible
2. After last wash, remove as much DPBS as possible by tilting plate back
3. For a 6 well plate, pipet 300 l of NP-40 buffer directly onto the cells. Make sure buffer
completely covers cells.
4. Keep plates on ice for 20 minutes, making sure that they are as flat as possible
5. Scrape the cells and collect the lysate in cold, labeled microcentrifuge tubes.
6. Spin samples at maximum speed for 15 minutes at 4°C.
7. Transfer the supernatant to new labeled cold microcentrifuge tubes
8. Store the samples (and the extra NP40 buffer for the BSA assay) at -80C.
Updated 03/04/14 by CM