(9c) Simultaneous High-Resolution Separation and Concentration of Proteins Using Microscale Pore Limit Electrophoresis

Here we present our recent work in photopolymerizing precise and well-controlled polyacrylamide porosity gradients in microchannels for microscale pore limit electrophoresis (μPLE) of native proteins. Pore limit electrophoresis was introduced in the late 1960's as a slab gel-based separation technique in which a mixture of proteins is fractionated along a polymer gradient of decreasing pore sizes reaching dimensions smaller than the effective molecular diameter of analyte species. In this manner, PLE provides high-resolution fractionation of complex samples based on the spatial dependence of each species' electrophoretic pore limit - the porosity at which a protein's electrophoretic mobility is negligible due to its molecular size. In μPLE, we aim to advance this separation technique by achieving certain advantages-of-scale associated with miniaturizing pore gradient gels, including faster run times and better reproducibility. The pore gradient gel is also useful for estimating size-exclusion membrane thresholds for a broad range of polymer concentrations and protein sizes simultaneously.

We adapted our previously-developed polymer gradient photopolymerization technique [1] to fabricate on-chip pore gradient gels. Porosity was controlled via distributions of acrylamide monomer and bisacrylamide crosslinker. We demonstrate simultaneous separation and concentration of proteins ranging in molecular weight from 21.5kDa ? 144kDa along 5mm μPLE gels spanning 10%T, 2.6%C ? 40%T, 12%C. Results show increasing resolution as proteins approach their effective pore limits. However, continued migration beyond this point shows electrophoretic behavior, including increased dispersion, which strays from theoretical predictions based on the Ferguson relationship. We will discuss potential explanations for the electrokinetic phenomena observed in this non-Ferguson regime. We will also show that μPLE can be used to concentrate dilute samples by exploiting the stacking phenomenon associated with an analyte's decreasing electrophoretic mobility. Concentration factors > 40,000 are easily achieved with dilute (100pM) samples.

[1] G.J. Sommer, A.K. Singh, A.V. Hatch. ?On-Chip Isoelectric Focusing Using Photopolymerized Immobilized pH Gradients? Anal. Chem., 2008, 80, 3327-3333.