Miniaturization of Protein Separation: Isoelectric Focusing and SDS-Page

Abstract

We have developed isoelectric focusing(IEF) and sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis(PAGE) techniques in very short channels (< 5 mm) that require less than 30 s to perform. The methods and chip architecture for these separations have been selected specifically for the eventual goal of their integration into two-dimensional(2D) protein separation systems. IEF of proteins was achieved with commercially available ampholytes in short microchannels (4 mm) fabricated in either polydimethylsiloxane(PDMS) or glass substrates. Detection was performed by fluorescence imaging of the channel using a CCD. We also prepared glass microchips for SDS-PAGE by photopolymerizing and photopatterning Polyacrylamide gel in microchannels. A protein marker sample (20.1–205kD) was analysed in less than 30 s in an effective length of ~ 2 mm, and the band movement was monitored by video microscopy. Minimizing the length of separation channel for the two separation methods leads to a number of improvements. 1) Separation of protein peaks could be achieved very quickly, typically within 30s. 2) The electric potential to be applied across the channel is greatly decreased, an important factor in design of integrated, portable systems. 3) Instead of mobilizing or eluting focused peaks, a low-magnfication microscope objective can be used for imaging of the channel in real time for a faster analysis. 4) The overall size of chip could be decreased significantly, thereby increasing the number of devices that can be fabricated on a wafer.