Focused strips were equilibrated for 15 min in equilibration buffer (6 M urea [Plus-One; GE Healthcare], 2% SDS, 30% glycerol, 0.05 M Tris [pH 8.8], 0.002% bromophenol blue) containing 1% DTT followed by a second equilibration for 15 minutes in equilibration buffer plus 4% iodoacetamide.
Strips were equilibrated in 6 M urea, 2% SDS, 30% glycerol, 0.375 M Tris-HCl pH 8.8, 0.002% bromophenol blue and 10 mg/ml DTT for 15 min, followed by a second equilibration step with the same buffer containing 25 mg/ml iodoacetamide instead of DTT, also for 15 min. Strips were loaded on 12% SDS-gels with an overlay of agarose solution (0,5 ml/100 ml electrophoresis buffer).
The strips were then equilibrated in SDS equilibration buffer containing 6 M urea, 75 mM Tris-HCl, pH 8.8, 29.3% v/v glycerol, 2% w/v SDS, 0.002% w/v bromophenol blue, and 1% w/v dithiothreitol (DTT) for 15 min, followed by a second equilibration using the same buffer containing 4.5% w/v iodoacetamide instead of DTT for another 15 min.
Prior to SDS-PAGE IPG-strips were equilibrated at room temperature in 6 M urea, 0.375 M Tris HCl pH 8.8, 2% SDS, 20% glycerol, 2% DTT for 10 min followed by a second equilibration in the same buffer except that DTT was replaced by 2.5% iodoacetamide.
The focused IPG strips were equilibrated at room temperature in buffer (50 mM Tris HCl pH 8.8, 6 M urea, 20% glycerol, and 2% SDS) with 2% DTT (w/v) for 15 min followed by a second equilibration in the same buffer containing 2.5% iodoacetamide (w/v) for 15 min.
The focused IPG strip was equilibrated with an equilibration buffer (6 M urea, 130 mM DTT, 112 mM Tris HCl pH 8.8, 4 % SDS, 30%% glycerol and 0.002 % bromophenol blue) for 15 min, followed by a second equilibration for 15 min in the same solution containing 135 mM iodoacetamide instead of DTT.
A second equilibration step was then performed with 2.5% (w/v) iodoacetamide added to the SDS equilibration buffer.
A second equilibration step was then performed in the same SDS equilibration buffer containing 250 mM iodoacetamide instead of DTT.
The final determination was performed between minutes 30 and 45 following a second equilibration interval.
A second equilibration step using the same reagents as above, but the DTT was replaced with 135 mM iodoacetamide.
After IEF the strips were equilibrated for 15 min in 50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 1% (w/v) SDS supplemented with 65 mM DTT, followed by a second 15 min equilibration in the same buffer containing 240 mM iodoacetamide instead of DTT.
