Enrichment and fractionation of proteins via microscale pore limit electrophoresis

In this work we photopolymerized precise and well-controlled polyacrylamide porosity gradients in microchannels for microscale pore limit electrophoresis (microPLE) of proteins. Porosity was controlled via distributions of acrylamide monomer and bisacrylamide crosslinker. MicroPLE 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. Proteins ranging in molecular weight from 21.5 kDa-144 kDa were separated under native buffering conditions along 5-mm- and 7-mm-long microPLE gels spanning 10%T, 2.6%C-40%T, 12%C. The pore gradient gel is useful for estimating size-exclusion thresholds for a broad range of polymer concentrations and protein sizes simultaneously. We show that microPLE can be used to concentrate dilute samples by exploiting the stacking phenomenon associated with an analyte's decreasing electrophoretic mobility. Concentration factors>40,000 were demonstrated with dilute (100 pM) samples. A detailed theoretical analysis of microPLE transport behavior based on Ferguson assumptions provides scaling and design parameters with which to tailor gels based on fractionation or enrichment needs. Experimental results show that the Ferguson assumptions break down as proteins migrate beyond an effective pore limit, prompting the need for further investigation into this non-Ferguson regime.