Digitonin Extraction Buffer for Differential Detergent Lysis

Recipe

Reagent	Per 100 mL of solution	Final concentration
PIPES buffer (4×)	25 mL	1×
Digitonin	18.75 mg	0.015%, pH 6.8 at 4°C
PMSF (100 mm)	1 mL	1 mm
EDTA (0.5 m)	0.6 mL	3 mm
ddH₂O	Add to 100 mL
Dissolve digitonin by adding to 10 mL of 4× PIPES buffer and boiling. Add PMSF slowly to the warm digitonin solution. Add the remaining 4× PIPES buffer, EDTA, and ddH₂O. Cool to 4°C and adjust pH to 6.8 with dilute HCl, and add ddH₂O to 100 mL. The final stock solution can be aliquoted and stored at −80°C. Thaw on ice before immediate use.