Cell Lysis Buffer (10X) #9803

1. If buffer will be continually used, it is recommended that the 10x buffer be kept at 4°C for 1-2 weeks. For longer periods of time, buffer should be stored at –20°C. Aliquoting of 10x buffer is recommended if many small experiments are to be performed.

2. Thaw 10x buffer at 24-30°C, mixing end-over-end.

3. Dilute 10X Cell Lysis Buffer to a 1X solution using ddH2O. This product supplies enough 10X material to make 150mls of whole cell extract.

4. Chill 1X buffer on ice and add PMSF just prior to use.

Note: CST recommends adding 1 mM PMSF immediately before use.

Lysis:

For lysis of adherent cells, we recommend the following: (all reagents and lysates must be kept cold)

1. Treat cells as desired.

2. Wash plate with PBS to remove residual media.

3. Add 400 µL of 1x lysis buffer/ 10 cm dish.

4. Incubate plate on ice for 5 minutes.

5. Scrape cells.

6. Sonicate briefly.

7. Spin extract 10 minutes at 14,000 x g in a cold microfuge.

8. Remove supernatant for use.

Additional notes:

1. For non-adherent cells, add 400 µl of buffer per 10⁷ cells once they have been washed in 1X PBS and pelleted.

2. 2X #9803 Cell Lysis Buffer can be used for lysis of tissue samples, although a homogenization step is recommended after adding lysis buffer. Extract the tissue at a

ratio of 100 mg of tissue to 1 ml of buffer. Sonication of the tissue lysate is also required.

3. Additional protease inhibitors can be added to the 1x lysis buffer without any difficulties.