After the IEF run, IEF gels were equilibrated in equilibration buffer (62.5 mM Tris HCl pH 6.8, 2.5% SDS, 10% (v/v) glycerol and 5% (v/v) 2-mercaptoethanol for 15 min twice.
Strips were equilibrated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and a trace of bromophenol blue) containing 10 mg/mL DTT for 15 min and then in equilibration buffer containing 25 mg/mL iodoacetamide for 15 min, and sealed to 12% acrylamide gels made in-house using 0.5% agarose in standard Tris-glycine electrophoresis buffer.
Subsequently the strips were equilibrated in equilibration buffer (50 mM TrisHCl, pH 8.8, 6 M Urea, 20% glycerol, 2% SDS) containing 1.5% DTT for 15 minutes, followed by 4% IAA in equilibration buffer for 15 minutes.
Afterwards, the strips were equilibrated consecutively in equilibration buffer [75 mM Tris/HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and 0.002% Bromophenol Blue] containing 10 mg/ml DTT and in equilibration buffer containing 25 mg/ml iodacetamide for 20 min each.
After IEF, the IPG strips were equilibrated sequentially, first in equilibration buffer (6 M urea, 0.375 M Tris-HCl pH 8.8, 2% SDS, and 20% glycerol) containing 2% dithiothreitol, then in equilibration buffer containing 2.5% iodoacetamide.
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.
For the second dimension, the IPG strip was equilibrated for 15 minutes in equilibration buffer containing DTT 2% w/v and then for 15 minutes in equilibration buffer containing iodoacetamide 2.5% w/v (equilibration buffer consisted of urea 6 M, glycerol 30% v/v, SDS 2% w/v, and 50 mM Tris/HCl pH 8.8).
Then the IPG strips were first equilibrated for 10 min in equilibration buffer 1 (6 M urea, 0.375 M Tris HCl pH 8.8, 20% glycerol, 2% SDS, 2% DTT) and subsequently in equilibration buffer 2 (6 M urea, 0.375 M Tris HCl pH 8.8, 20% glycerol, 2% SDS, 2.5% iodoacetoamide) for 10 min.
Afterwards, IPG strips were equilibrated for 15 min in equilibration buffer (50 mM Tris, pH 8.8, 6 M urea, 2% (w/v) SDS, 30% glycerol and 0.04% (w/v) bromophenol blue and then in equilibration buffer containing 10 mg-1 DTT for 15 min.
After IEF, the strips were equilibrated for 15 min in equilibration solution (0.05 M Tris, 6 M urea, 30% glycerol, 2% SDS, pH 8.8) containing 65 mM DTT followed by 15 min in equilibration solution containing 135 mM iodoacetamide to block free thiol groups.
Then strips were equilibrated for 15 min in equilibration solution (6 urea, 50 m Tris-HCl (pH 8.8), 30% glycerol, 2% sodium dodecyl sulphate) containing 10 mg ml−1 dithiothreitol (Roche Diagnostics, Mannheim, Germany) and subsequently for 15 min in equilibration solution containing 25 mg ml−1 iodoacetamide (Sigma-Aldrich, St Louis, MO, USA).
