Dear Lauriane,

Instead of lysis buffer you can extract the cells using 95% methanol pH 2. and incubate overnight at 4 C with stirring. Then you can centrifuge to remove the cells and the supernatant can be evaporated 10 folds using rotary evaporator to remove the methanol. The resulted aquas phase is the total protein extract of the cells. For cells obtained from 1 L culture you can use 200 mL of the 95% methanol pH2. You can also use Acetonitrile (ACN 60%) or isopropanol 70% pH2. This method is compatible with MALDI-TOF analysis for bacterial or cells identification and total protein analysis.

For Western blotting I wash in 20mM Tris pH 8.0, 5mM EDTA then sonicate in 20mM Tris pH 8.0, 5mM EDTA + protease inhibitor. Always works great for me. Depends on what you want to use the total protein for I guess...

Dear Lauriane

I have used RIPA buffer to extract total protein (Cytoplasm+nucleoplasm) from MEFs which is an embryonic cell line. It works always well for me! :) 

I think the lysis buffer (20mM Tris-HCl (pH 7.4), 140mM NaCl, 5mM MgCl2 and 1% Triton X-100) is very good

Hi,

Dear Lauriane , I send you the method of my paper that we experienced in bacteriology lab for range of gram positive bacteria including Streptomyces spp., for several times.

The method for negatives is different.

For extraction of Gram-positive bacteria protein, extraction buffer with 100 ml volume of sterile distilled water was prepared as follows.

Tris 0.75 g 5 ml of beta-mercaptoethanol Glycerol 13 ml EDTA 9/1 pH 8/6

To perform the extraction process, the strains were suspended in 1 ml of sterile distilled water after 25 days of growth on NA at 25-28 ° C. The optical absorption of the bacterial suspension was adjusted to 600 nm by one. 20 microliters of lysozyme was added after incubation for 30 minutes at 37 ° C, 100 µL of 20% sodium dodecyl sulfate was added to it(suspention) and boiled for 10 minutes in boiling water. Finally, the samples were centrifuged at a centrifuge at 10,000 rpm for ten minutes and the clear supernatant was stored as a protein in the freezer at -20 ° C.

Be sure with that for gram + bacteria strains which I extracted...

Good luck with your work