I am using an adherent cancer cell line and I need a whole cell lysis buffer for protein isolation. Can anyone tell the composition of the whole cell lysis buffer?
Once prepared lysis buffer may be stored at 4°C for several weeks or for up to a year aliquoted and stored at -20°C.
Composition:
150 mM NaCl
1.0% NP-40 (possible to substitute with 0.1% Triton X-100)
50 mM Tris-HCl pH 8.0
Protease Inhibitors (combination of PMSF, leupeptin, Aprotinin, etc.)
Preparation of lysate from cell culture:
1. Place the cell culture dish in ice and wash the cells with ice-cold PBS.
2. Aspirate the PBS, then add ice-cold lysis buffer (1 ml per 10^7 cells/100 mm dish/150 cm^2 flask; 0.5ml per 5x10^6 cells / 60 mm dish / 75 cm^2 flask).
3. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube.
4. Maintain constant agitation for 30 minutes at 4°C.
5. Spin at 16,000 x g for 20 minutes in a 4°C pre-cooled centrifuge.
6. Gently remove the tube from the centrifuge and place on ice. Transfer the supernatant to a fresh tube kept on ice, and discard the pellet.
Hope this helps.
Hi,
We use two buffers for whole cell extracts, depending on what do you need your extracts for.
RIPA - very strong, can even extract membrane proteins, but extracts are not suitable for subsequent IP (but you can easily use them for western blotting). Recipe: 150mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40 dissolved in 50mM Tris (pH 8.0).
The other buffer we just call lysis buffer: 300mM NaCl, 50mM HEPES pH7.6, autoclave and add Triton X-100 to 1%.
Just before use we add to each protease inhibitors. Personally, if I don't need to do IP, I prefer to use RIPA.
Hope that helps.
The Laemmli SDS-PAGE sample buffer would be the benchmark total protein extraction buffer. Depending on your downstream applications you may or may not need to modify the extraction method.
Once prepared lysis buffer may be stored at 4°C for several weeks or for up to a year aliquoted and stored at -20°C.
Composition:
150 mM NaCl
1.0% NP-40 (possible to substitute with 0.1% Triton X-100)
50 mM Tris-HCl pH 8.0
Protease Inhibitors (combination of PMSF, leupeptin, Aprotinin, etc.)
Preparation of lysate from cell culture:
1. Place the cell culture dish in ice and wash the cells with ice-cold PBS.
2. Aspirate the PBS, then add ice-cold lysis buffer (1 ml per 10^7 cells/100 mm dish/150 cm^2 flask; 0.5ml per 5x10^6 cells / 60 mm dish / 75 cm^2 flask).
3. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube.
4. Maintain constant agitation for 30 minutes at 4°C.
5. Spin at 16,000 x g for 20 minutes in a 4°C pre-cooled centrifuge.
6. Gently remove the tube from the centrifuge and place on ice. Transfer the supernatant to a fresh tube kept on ice, and discard the pellet.
Hope this helps.
There are several answers to this question based upon what your down stream application is, stability of the protein of interest, etc. For Co-IP experiments, we routinely used the NE-PER kit from Pierce that has all the buffers you would need to extract nuclear and cytoplasmic fractions from tissue culture cells. I have also used the following recipes for Co-IP using whole cell lysis in cell culture cells:
Molk Lysis: Final concentrations provided:
20mM NaPO4 pH7
150mM NaCl
2mM MgCl2
1% NP40
10% glycerol
10 mM NaF
0.1mM Sodium Orthovanadate
10mM Sodium Pyrophosphate
1mM DTT
1 Roche Complete protease inhibitor tablet (for 50mls of buffer)
stable 1-2 weeks at 4C or 12 weeks at -20
Funato Lysis:
50mM Tris-HCl pH7.4
150 mM NaCl
1mM EDTA pH8
1% TritonX-100
1 Roche complete protease inhibitor tab (for 50ml of water)
Mike's Special Lysis:
25mM Tris pH7.4
150mM NaCl
1% TritonX-100
1mM EDTA pH 8
1mM DTT
10mM NaF
Above stable at 4C for 6 months
Add just prior to use Roche Complete inhibitor stock and 1mM PMSF
For Luciferase experiments, we used the Passive Lysis Buffer provided with the Promega kit.
If you are just wanting to lyse the cells and look for presence or absence of the protein of interest by Western blot, then any of the above lysis buffers will work. I have successfully done a Bradford Assay (Bio-Rad) using a small amount of clarified lysate from all three of the above methods.
My last bit of advice would be to do a literature search for the protein you are interested in, and see what they successfully used with the downstream application you are going for.
Good luck and hope a little of this helps!
It all depends upn your purpose. Say if you really want to study proteins that may localized in nucleus then RIPA is the the best. However, RIPA is very powerful and will give you sticky DNA that can make obtaining the supernatant difficult. Lysates from RIPA can cmmonly give me wedges in electrophoresis. I have changed to MOPS buffer which so far provide me a better separation and less dirty background when nuclera proteins are not my focus.
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