Cell Lysis - (Dec/19/2008 )

I am taking expression in BL21 DE3 cells. If i have to run the SDS gel whats the method to lyse the cell just to check the soluble expression of the protein.....
I have sonicator in my lab......

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My method is:

A pellet from a 50 ml expression culture resuspended in 5 ml of lysis buffer (20 mM Tris-HCl (pH 7.5), 250 mM NaCl, 1% Triton X-100), followed by sonication for 6 X 15 seconds with 1 minute intervals. Spin the lysate at 13,000 rpm for 20 minutes and remove the supernatant (soluble fraction). Resuspend the pellet (insoluble fraction) in 5 ml lysis buffer. Analyse both the soluble and insoluble fraction by SDS-PAGE.

If your expression is low you can probably get away with resuspending your pellets in 2 ml.

P

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Triton x-100 and NP40 are common lysis buffers which are also components of RIPA buffer, but there are many other lysis buffers.

CHAPS buffer saves conformation of many proeins.
Digitonin also saves conformation but as long as it is a mild detergent only breaks cell membrane, not nuclei.

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