Freeze-thaw lysis buffer

20 ml KCl (3 M)

2 ml Tris-Cl (1 M, pH 7.8)

20 ml glycerol

1 ml Pefabloc (40 mg/ml)

100 μl leupeptin (10 mg/ml)

500 μl pepstatin (2 mg/ml)

50 μl aprotinin (10 mg/ml)

Add H₂O to make a final volume of 100 ml. Add Pefabloc, leupeptin, pepstatin, and aprotinin fresh each time. The final concentrations should be 600 mM KCl, 20 mM Tris-Cl (pH 7.8), 20% glycerol, 0.4 mg/ml Pefabloc, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 5 μg/ml aprotinin.