Table 3. Commonly used lysis buffers for lysing cultured cells

Buffer	Comments
NP-40 lysis	This is probably the most widely used lysis buffer. It relies on the nonionic detergent NP-40 as the major solubilizing agent, which can be replaced by Triton X-100 with similar results. Variations include lowering the detergent concentration or using alternate detergents such as digitonin, saponin, or CHAPS.
150 mM NaCl
1.0% Nonidet P-40 (NP-40)
50 mM Tris-Cl (pH 7.4)

RIPA lysis	This is a much harsher denaturing lysis buffer than NP-40, owing to the inclusion of two ionic detergents (sodium dodecyl sulfate [SDS] and sodium deoxycholate). In addition to releasing most proteins from cultured cells, RIPA lysis buffer disrupts most weak noncovalent protein-protein interactions.
150 mM NaCl
1.0% Nonidet P-40 (NP-40)
0.5% sodium deoxycholate
0.1% SDS
50 mM Tris-Cl (pH 7.4)

When studying the modification of proteins by phosphorylation, phosphatase inhibitors (e.g., 25 mM sodium fluoride [NaF], 40 mM β-glycerol phosphate, 100 μM sodium orthovanadate [Na₃VO₄], or 1 μM microcystin) should be included. If proteolytic degradation of the target protein is a problem, protease inhibitors should be included in the lysis buffer. Some commonly used inhibitors include aprotinin (1 μg/mL), leupeptin (1 μg/mL), pepstatin (1 μg/mL), and phenylmethylsulfonyl fluoride (PMSF; 50 μg/mL). Alternatively, commercially available protease cocktail tablets (e.g., Boehringer) can be included.