Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation

Abstract

Isoelectric Focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly (vinyl) alcohol (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370V/cm) efficient cooling maintains solution temperature inside the separation channel controllably in the range 2 -25 °C. Protein samples with a dynamic concentration range between µg/mL to mg/mL can be loaded into the micro device at a flow rate of 1 mL/hr and residence time of ~12 min. By using a protein complex of 9 proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device to device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10 to 20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The micro device has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches.

Flocculation on a chip: a novel screening approach to determine floc growth rates and select flocculating agents

“…[33][34][35][36] These systems have been used extensively to study upstream bioprocessing steps, [37][38][39][40][41] but the number of reports of microfluidic devices developed to study process optimisation of downstream processing steps, such as purification and separation of biomolecules, is still limited. 27,[42][43][44][45][46][47][48] In downstream bioprocessing, flocculation has attracted renewed interest but suitable analytical tools to comprehensively understand the flocculation mechanism have been absent. The precise control over the microenvironment afforded by microfluidic devices opened up an opportunity to investigate, for the first time, the formation and growth of flocs independently from floc breakage and ageing phases.…”

Advances in steady-state continuous-flow purification by small-scale free-flow electrophoresis

“…A similar cooling set-up was also incorporated in µFFE devices [49]. More recently, µFFE devices employed thermoelectric cooling plates, which can remove heat even faster [50].…”

Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis

“…Despite recent advances in μIEF techniques for biological analyses (Das et al, 2007;Sommer and Hatch, 2009;Wen et al, 2010), many attempts have focused on the easy separation of complex biomolecules according to the average pIs. To the best of our knowledge, there have been no attempts in integrating the μIEF technique for the simultaneous detection of cascade phosphorylation.…”