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.
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.
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.
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 leupeptinNote: CST recommends adding 1 mM PMSF immediately before use.
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.