Horizontal SDS-PAGE for IPG-Dalt

After IEF-IPG (see Chapters 21 –24 ), the IPG gel strips are equilibrated in the presence of SDS, DTT, urea, glycerol, and iodoacetamide, and placed onto the surface of a horizontal or on top of a vertical SDS gel (1 –3 ) (see Chapter 26 ). For horizontal setups, the laboratory-made or ready-made SDS-PAGE gel (ExcelGel SDS), cast on plastic backing, is placed onto the cooling plate of a horizontal electrophoresis system. The equilibrated IPG gel strip(s) is (are) transferred gel-side down onto the surface of the SDS gel alongside the cathodic electrode wick. For vertical setups, the equilibrated IPG gel strips are loaded on top of vertical SDS polyacrylamide gels and embedded in agarose (see Chapter 26 ). On completion of electrophoresis, the resolved polypeptides are either stained with Coomassie blue or silver nitrate, or are transferred onto an immobilizing membrane and detected with specific reagents, such as lectins or antibodies. Alternatively, they can be subjected to amino acid (sequence) analysis or mass spectrometry (4 –8 ).