Alternative Two Dimensional Electrophoresis – OFFGEL Electrophoresis Combined with High Sensitivity Microfluidic on-Chip Protein Detection

Abstract

Two dimensional gel electrophoresis (2D-GE) employs isoelectric focusing in the first dimension and a separation of the proteins according to their molecular weight in the second dimension. The gels are then stained using silver stain to visualize the protein pattern. This method is unrivalled in terms of resolution but is a tedious and time-consuming procedure. Here we present a combination of two easy methods that separate proteins in analogy to 2D-GE according to their isoelectric point (pI) and molecular weight (kDa). For the first dimension, OFFGEL electrophoresis was used. This newly developed method takes advantage of the impressive resolving power of immobilized pH gradient gel based isoelectric focusing (IPG IEF) but in contrast to conventional isoelectric focusing delivers sample in liquid phase thus avoiding sample recovery from the gel. For the second dimension, a microfluidic high sensitivity on-chip protein sizing method was employed. This method allows separating proteins from 5 to 250 kDa and offers a sensitivity equivalent or better than silver staining and a linear dynamic range across four orders of magnitude. Our data demonstrates that it is possible to easily detect a 1 % change in protein expression.