Gel-free IEF in a membrane-sealed multicompartment cell for proteome prefractionation.
A minidevice for performing gel-free proteome prefractionation via conventional IEF in soluble carrier ampholyte buffers is reported here. It consists of a compact block of polyoxymethylene in which eight samples and two electrode chambers are machined. Each of the eight sample chambers can be filled with up to 120 microL of sample and has the following size: 7 mm width, 3 mm depth and 10 mm height. The anodic and cathodic compartments have the same width and height as the sample chambers, but with a depth of 6 mm, thus accepting up to 250 microL of electrodic solutions. Focusing is in general accomplished in 2 h with a voltage gradient of up to 1000 V (7 cm electrode distance). Easy fractionation and collection of the content of the eight chambers is achieved by simply pressing a rubber diaphragm against the edges of the thin walls separating each well, this automatically breaking liquid continuity. The performance of this device has been tested by subfractionating total cell lysates of a human cancer cell line (U2Os) and of Escherichia coli bacterial cells, and by analysing the content of each chamber by mono-dimensional SDS-PAGE and 2-D maps.
Lam
Hoang-Trang
HT
Laboratoire d'Electrochimie Physique et Analytique, EPFL SB ISIC LEPA, Lausanne, Switzerland.
Antonioli
Paolo
P
Righetti
Pier Giorgio
PG
Citterio
Attilio
A
Girault
Hubert
H
D001426:Bacterial Proteins / Q000032:analysis; D002457:Cell Extracts / Q000737:chemistry; D005591:Chemical Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D005782:Gels; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation* / Q000379:methods*; D007526:Isoelectric Point; D009363:Neoplasm Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D002457:Cell Extracts; D005782:Gels; D009363:Neoplasm Proteins; D020543:Proteome