Description
Product Usage Information
1. Thaw 10x buffer at 24-30°C, mixing end-over-end. 2. Dilute 10X Cell Lysis Buffer to a 1X solution using ddH2O. This product supplies enough 10X material to make 150mls of whole cell extract. 3. Chill 1X buffer on ice and add PMSF just prior to use. Lysis: All reagents and lysates must be kept cold. 1. Treat cells as desired. 2. Wash plate with PBS to remove residual media. 3. Add 1X to cells: (a) For adherent cells, add 400 µl of 1X Cell Lysis buffer/10 cm dish. (b) For non-adherent cells, add 400 µl of buffer per 10 7 cells once they have been washed in 1X PBS and pelleted. ââââ ââ 4. Incubate plate on ice for 5 minutes. 5. Scrape cells. 6. Sonicate briefly. 7. Centrifuge extract for 5 minutes at 14,000 x g in a cold microfuge. 8. Remove supernatant for use. 1X 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. Additional protease inhibitors can be added to the 1X lysis buffer without any difficulties.
Storage
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 For long term storage, keep 10X buffer at -20°C. If buffer will be continually used, it is recommended that the 10X buffer be kept at 4°C for 1-2 weeks. Aliquoting of 10X buffer is recommended if many small experiments are to be performed. 1X solution can also be aliquotted into single use aliquots and stored at -20°C.
