Ipg dry strips

Sephacryl S-300 fractionated NE (200 g) was concentrated by using Microspin G-25 columns (Amersham Biosciences, USA) and resuspended in rehydration solution (8 M urea, 4% CHAPS, 0.5% IPG buffer, 2 mM tributyl phosphine) followed by IEF analysis as described previously (Woo et al., 2002) . Three successive concentration and reconstitution cycles in rehydration solution was followed for buffer exchange and removal of salts for IEF.
The samples (250 l) were then loaded onto IPG Drystrips (13 cm, pH 3-10L or 4-7L;
Amersham Biosciences, USA) and loaded onto an IPGphor (Amersham Biosciences, USA)
A c c e p t e d M a n u s c r i p t 8 before commencing the following protocol: 12 h rehydration, 100 V for 100 Vh, 250 V for 250 Vh, 500 V for 1000 Vh, 1000 V for 2000 Vh, 8000 V to 60 000 Vh. Following IEF, strips were rocked in urea free equilibration solution (50 mM tris, 30% glycerol (v/v), 2% SDS (w/v), 2 mM tributyl phosphine) for 30 minutes. IPG strips were dissected uniformly and diced into 2 volumes of renaturation buffer. The nuclear proteins eluted from each gel slices were recovered and assayed for DNA binding activity in EMSA. The pI of each gel piece was determined from the supplied standard curve (Amersham Bioscience).