Cell lysis is the breaking down of the cell membrane and the separation of proteins from the non-soluble parts of the cell. Lysate buffers contain different detergents that help to release soluble proteins (Triton-X, Tween, SDS, CHAPS). Dependent on the location of the protein of interest, a different lysate buffer is needed to obtain a high yield and purity of the protein.
Pre-cool a refrigerated centrifuge to 4°C. Pellet the cultured cells by centrifugation for 5 minutes at 1000 x g (approximately 2000 rpm) at 4°C. Wash 3 times with ice-cold 1X PBS and then add chilled RIPA buffer with protease inhibitor. In general, add 100μl RIPA buffer for approximately every 106 cells present in the pellet (count cells before centrifugation). Reduce the volume of RIPA buffer accordingly if a higher protein concentration is required. Vortex to mix and keep on ice for 30 min, vortexing occasionally.
Dissect the tissue of interest and wash briefly with chilled 1X PBS to remove any blood if necessary, cut the tissue into smaller pieces whilst keeping it on ice. Transfer the tissue to a homogenizer and add RIPA buffer with protease inhibitor. In general, add 500μl RIPA buffer for approximately every 10 mg of tissue. Homogenize thoroughly and keep the sample on ice for 30 min. Vortex occasionally.
Tip 1: Add phosphatase inhibitors to lysis buffers for extraction of phosphorylated proteins.
Sonicate the sample to break the cells or tissue up further and to shear DNA. Adjust sonication time to your type of sample: 1 min for cell lysates and 2–5 min for tissue lysates at a power of about 180 watts (in rounds of 10 seconds sonication/10 seconds rest for each cycle). Keep the sample on ice during the sonication.
Tip 2: The addition of DNase for DNA digestion is not recommended as this introduces protein contamination from the enzyme.
|1X PBS||For 1000 ml|
|10 mM Na₂HPO₄||1.42 g|
|1.8 mM NaH₂PO₄||0.24 g|
|137 mM NaCl||8 g|
|2.7 mM KCl||0.2 g|
|Adjust pH to 7.4|
|Add ddH₂O to 1000 ml|
|RIPA buffer||For 1000 ml|
|50 mM Tris•HCl, pH 7.4||50 ml|
|150 mM NaCl||8.76 g|
|1% Triton X-100 or NP-40||10 ml|
|0.5% Sodium deoxylcholate||5 g|
|0.1 % SDS||1 g|
|1 mM EDTA (0.5 M stock)||2 ml|
|10 mM NaF||0.42 g|
|Add ddH₂O to 1000 ml|
|Add PMSF to a final concentration of 1 mM and any other protease inhibitors immediately before use.|
|4X SDS sample buffer||For 1000 ml|
|12% SDS||120 g|
|25% Glycerol||250 ml|
|150 mM Tris•HCl (pH 7.0•1M stock)||150 ml|
|0.03% Bromophenol Blue||300 mg|
|20% β-mercaptoethanol||200 ml|
|Add ddH₂O to 50 ml, aliquot and store at -20°C|
|20% β -mercaptoethanol, (or 500 mM DTT replaced), should be added freshly before use.|
Cell and tissue lysate preparation
Downloadable PDF protocol