Bacterial Lysis for Protein Preparation

Dominik Haudenschild, November 08, 1994, from LJCRF

Method

1. Spin 50ml culture in 50ml tube for 10 minutes at 10,000g

2. Resuspend pellet in 1.6ml TEN with PI

3. Transfer pellet to 12x75 falcon tube

4. Add 200µl 10mg/ml Lysozyme

5. Incubate 10 minutes on ice

6. Add 25 µl of 10% NP-40

7. Incubate 10 minutes on Ice

8. Sonicate with bursts on ice

9. Transfer to 2ml microcentrifuge tubes

10. Spin for 10 minutes

11. Keep Supernatant, which contains protein.

Materials

TEN is 50mM Tris, 1mM EDTA, 100mM NaCl pH 7.5

PI's are Aprotinin, 2µg/ml
Leupeptin, 0.5µg/ml
Pepstatin, 0.7µg/ml
EDTA, 2mM