Urea lysis buffer
2% (w/v) CHAPS
2% (v/v) carrier ampholytes (pH 3-10)
10 mM Pefabloc proteinase inhibitor
To prepare 50 ml of urea lysis buffer, dissolve 30.0 g of urea in deionized H2O and adjust the volume to 50 ml. Add 0.5 g of Serdolit MB-1, stir for 10 minutes, and filter. Add 1.0 g of CHAPS, 0.5 g of DTT, and 1.0 ml of Pharmalyte (pH range 3-10) to 48 ml of the filtered urea solution. If necessary, add 50 mg of Pefabloc proteinase inhibitor immediately before use.
IMPORTANT:
Lysis buffer must be freshly prepared. Alternatively, make 1-ml aliquots and store at -80ºC for up to several months. Do not refreeze lysis buffer that has already been thawed! Never heat urea solutions above 37ºC. Otherwise, protein carbamylation may occur!
