Directions for Use

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. Aliquotting 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 buffer in Milli-Q water or water of equivalent quality.
4. Chill 1x buffer on ice and add PMSF just prior to 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 ul of 1x lysis buffer/10 cm dish.
4. Incubate plate on ice for 5 minutes.
5. Scrape cells.
6. Sonicate briefly.
7. Centrifuge extract for 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 ul of buffer per 10⁷ cells once they have been washed in 1X PBS and pelleted.
2. The CST 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 lysis buffer without any difficulties.

Solutions and Reagents

• 1X Cell Lysis Buffer:
• 20 mM Tris-HCl (pH 7.5)
• 150 mM NaCl
• 1 mM Na₂EDTA
• 1 mM EGTA
• 1% Triton
• 2.5 mM sodium pyrophosphate
• 1 mM beta-glycerophosphate
• 1 mM Na₃VO₄
• 1 µg/ml leupeptin

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

Application References

• Kong, B. et al. (2010) Oncogene 29, 5146-58. Applications: Western Blotting

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Companion Products

• 9802 Kinase Buffer (10X)
• 9806 RIPA Buffer (10X)