Contributor: Suprya JayadevDate: May 25, 1993

Reagents:

Lysis buffer25 mM Tris-HCl, pH 7.45 mM EDTA1 mM ATP20 μg/ml CLAP1 mM PMSF

Buffer A10 mM MgCl20.2 M Tris-HCl, pH 7.40.2 % Triton X-100

Buffer B0.2 M sodium acetate, pH 5.00.2 % Triton X-100

Buffer C0.2 M Tris-HCl, pH 7.40.2 % Triton X-100--------------------------------------------------------------------------------

Isolating cellular enzyme

1) Grow 5 x 108 cells under regular growth conditions.

2) Pellet cells and wash one time with ice cold PBS.

3) Resuspend pellet in 10 ml of ice cold lysis buffer.

4) Bomb cells: 20 minutes @ 350 psi, 4°C

5) Spin lysed cell mixture at 2,100 rpm, 4°C for 10 minute.

6) Discard pellet and set aside 3 ml of supernatant.

--> Supernatant from this step = "homogenate"

7) Spin remaining supernatant (" 7 ml) @ 200,000 xg,4 °C for 30 minutes.

centrifuge ____________________rotor ____________________rpm ____________________time @ plateau ____________________

8) Recover both supernatant and pellet separately.

--> Supernatant = "cytosol"

9) Resuspend pellet in 3 ml lysis buffer.

--> This fraction = "membrane"--------------------------------------------------------------------------------