An immobiline DryStrip application method enabling high-capacity two-dimensional gel electrophoresis.
In the field of proteomics the need to detect low-abundance cellular components, such as regulatory proteins, is of critical importance. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the most commonly used separation tools for these biological investigations. In this paper we report an alternative micropreparative 2-D PAGE sample application method, called the "paper bridge loading" method. This method makes it possible to apply a larger sample volume to commercially available immobilized pH gradient (IPG) strips. The Vh products required for focusing are only marginally longer than those used in analytical experiments. The method was compared to traditional cup loading and in-gel rehydration. With 18 cm long narrow-range Immobiline DryStrip pH 4.5-5.5, the "paper bridge" method allowed the application of 10 mg human plasma proteins compared to 3 mg with traditional loading methods. The corresponding figures using Escherichia coli sample 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 separation of high molecular weight proteins.
Karolinska Institutet, Department of Medicine, Karolinska Hospital, Stockholm, Sweden.
F
F
Sabounchi-Schütt
J
J
Aström
I
I
Olsson
A
A
Eklund
J
J
Grunewald
B
B
Bjellqvist
D000178:Acrylamides; D001426:Bacterial Proteins / Q000032:analysis; D001798:Blood Proteins / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans
D000178:Acrylamides; D001426:Bacterial Proteins; D001798:Blood Proteins; C059413:Immobiline