An Immobiline DryStrip application method enabling high-capacity two-dimensional gel electrophoresis

In the field of proteomics the need to detect low-abundance cellular compon ents, such as regulatory proteins, is of critical importance. Two-dimension al polyacrylamide gel electrophoresis (2-D PAGE) is one of the most commonl y used separation tools for these biological investigations. In this paper we report an alternative micropreparative 2-D PAGE sample application metho d, called the "paper bridge loading" method. This method makes it possible to apply a larger sample volume to commercially available immobilized pH gr adient (IPG) strips. The Vh products required for focusing are only margina lly longer than those used in analytical experiments. The method was compar ed to traditional cup loading and in-gel rehydration. With 18 cm long narro w-range Immobiline DryStrip pH 4.5-5.5, the "paper bridge" method allowed t he application of 10 mg human plasma proteins compared to 3 mg with traditi onal loading methods. The corresponding figures using Escherichia coli samp le was found to be 6 mg and less than 2 mg, respectively. The paper bridge method also showed the best results in terms of spot resolution and separat ion of high molecular weight proteins.