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 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.
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 supernant for use.
1. For non-adherent cells, add 400 µl of buffer per 107cells 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.
Solutions and Reagents:
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.
Storage:Store at -20°C. For short term storage (1-2 weeks), Cell Lysis Buffer can be stored at 4°C.