1. If buffer will be used regularly, it is recommended that the 1X buffer be kept at 4ºC for 1-2 weeks. For longer periods of time, buffer should be stored at -20ºC. Aliquotting of 1X buffer is recommended to avoid freeze thaw cycles.
2. Thaw 1X buffer at 24-30ºC, mixing end-over-end.
3. Chill 1X buffer on ice and add PMSF just prior to use.
Note: Cell Signaling Technology recommends adding 1 mM PMSF (final concentration) 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 ice-cold PBS to remove residual media.
3. Add 500–1000 µl of lysis buffer/ 10 cm dish.
4. Incubate plate on ice for 2 minutes.
5. Collect cell lysate solution for use. Spin extract 1 minute at 14,000 x g in a cold microfuge if cell debris is present.
1. For non-adherent cells, judgment must be used as to amount of lysis buffer to add. Addition of volume equal to cell pellet is generally appropriate.
2. Additional protease inhibitors can be added to the 1X lysis buffer if needed.
1X Lysis Buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton, 20 mM sodium Pyrophosphate, 25 mM Sodium Fluoride, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptinNote: Cell Signaling Technology recommends adding 1 mM PMSF immediately before use.
Store at -20°C. For short term storage (1-2 weeks), Cell Lysis Buffer can be stored at 4°C.
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