The protein powder was weighed and then resuspended
in lysis buffer [9 mol L-1 urea, 4% (w/v) CHAPS, 65
mmol L-1 DTT, and 0.5% (v/v) carrier ampholytes pH
3.5-10] and sonicated in ice bath for 20 min. After
centrifugation at 12 000 × g for 30 min, the supernatant
was collected. Protein concentration was determined
according to the method described by Bradford (1976).