11/8/2014 Simultaneous semi-dry electrophoretic transfer of a wide range of differently sized proteins for immunoblotting. : Protocol Exchange http://www.nature.com/protocolexchange/protocols/2925#/equipment 1/8 PROTOCOL EXCHANGE | COMMUNITY CONTRIBUTED Subject terms: Biochemistry Isolation, Purification and Separation Protein analysis Cell biology Keywords: Immunoblotting SDS-PAGE Gradient gel Phosphorylated eIF2 alpha Semi-dry protein transfer Simultaneous semi-dry electrophoretic transfer of a wide range of differently sized proteins for immunoblotting. Martin Wiedemann , Su Jung Lee , Richard Cardoso da Silva , Jyothsna Visweswaraiah , Josefin Soppert & Evelyn Sattlegger Sattlegger Lab Abstract Usage of a semi-dry electrophoretic apparatus is a fast and cost effective way to transfer proteins from polyacrylamide gels to membranes for immunoblotting. However, its efficiency is usually limited to small and mid-range molecular weight proteins, and the time-consuming wet transfer procedure is usually the preferred method for high molecular weight proteins. Here we describe a protocol for the efficient semi-dry transfer of proteins of diverse sizes (ranging from 15 to 296 kDa) resolved in a gradient SDS-PolyAcrylamide Gel Electrophoresis (PAGE) gel. This protocol was successfully employed to transfer and detect the small protein Rps22 (15 KDa), the high molecular weight protein Gcn1 (296 kDa), among others. This protocol is also suitable for the quantitative detection of phosphorylated proteins such as phosphorylated eIF2 alpha. Reagents Reagents for casting protein gels: • 40 % acrylamide (29:1). Dissolve 193.4 g acrylamide and 6.6 g bis-acrylamide in 500 ml dH2O. Store at 4°C, protected from light. • 1.5 M Tris-HCl (pH 8.8). • 10 % [w/v] SDS in dH2O. • 4 % acrylamide premix: 20 ml 40 % acrylamide (29:1), 51 ml 1.5 M Tris-HCl (pH 8.8), 2 ml 10 % SDS, 127 ml dH2O. Store at 4°C, protected from light. • 17 % acrylamide premix: 85 ml 40 % acrylamide (29:1), 51 ml 1.5 M Tris-HCl (pH 8.8), 2 ml 10 % SDS, 62 ml dH2O. Store at 4°C, protected from light.