IEF and cIEF Analysis
IEF was used to confirm the pI of proteins and was performed using a pH
3−10 IEF gel (Invitrogen, CA, USA) with 18 µg samples. Coomassie Blue
staining was used for visualization. To quantitate each pI band, we
performed cIEF using a neutral capillary (50 µm internal diameter; total
length, 30.2 cm; effective length, 20 cm) on a PA800 plus CE instrument
(Beckman Coulter, Brea, CA, USA). Samples (5 mg/mL, 10 µL) were prepared
and mixed with 240 µL of master mix, and 200 µL was loaded onto the
sample tray. UV detection was performed at 280 nm.