To achieve a good protein solubilisation in the first dimension, Urea, Ampholytes and detergents were added to the immobilized pH-gradient strips by rehydration.
The normally suggested IPG-strip rehydration-cocktail:
8 M Urea, 0.5% (w/v) Pharmalytes (also included in GE’s IPG-strip buffer) + additives.
....can cause a high background in the second dimension due to unsoluble SDS-Pharmalytes complexes on the
alkaline side of the gel. Use the additional washing steps described for 2D-gels in the different staining methods.
See the 2D trouble shooting “Blue Curtain”.
*Bromophenolblue **Bromophenolred (dyes), ***Bishydroxyethyldisulfide (Aldrich #38,047-4, for pH-gradients including basic region)
* if available