Cell Lysis Buffer - Non-denaturing

Description: Cell Lysis Buffer is used to lyse cells under non-denaturing conditions.

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 b-glycerophosphate, 1 mM Na3VO4 , (sodium ortho-vanadate),1µg/ml leupeptin

Alternative buffer: (10mM imidazole pH 7.3, 0.5 M NaCl, 1% Triton X-100, 0.2mM sodium ortho-vanadate, 0.2mM PMSF, 2mM sodium azide)

Note: We recommend adding the 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.

Cell Lysis

Non-Denaturing Conditions (by using boiling lysis buffer) Rinse adherent or non-adherent cells from a 15cm dish with an excess of phosphate-buffered saline (PBS); repeat wash. Lyse cells with 6ml of ice-cold lysis buffer with gentle rotation at 4C, 15-30 minutes. Remove the lysate from the plate and centrifuge at 40,000rpm for 1.5 hour at 4C. Collect supernatant and use. For IP dilute 1:1 with lysis buffer less triton X-100.

Optional Denaturing Conditions Rinse adherent or non-adherent cells from a 15cm dish with an excess of phosphate-buffered saline (PBS); repeat wash. Lyse cells with 3ml of boiling lysis buffer (10mM Tris pH 7.4 , 0.2mM sodium ortho-vanadate). Microwave the cells for 5 seconds to assure complete lysis and denaturation. Scrape the lysate from plates and centrifuge at 40,000rpm for 1.5 hour at 15C. Dilute the lysate 5-fold with lysis buffer. Adjust the lysate to final concentrations of 1.0% Triton X-100, 0.2% SDS, 10mM imidazole pH 7.3, 0.2mM sodium ortho-vanadate, 1mM EDTA.