Journal Article

Controlling the separation of native proteins with temperature in thermal gel transient isotachophoresis

Peli Thanthri, Shakila H., Linz, Thomas H.

2023

Overview

Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution. Thermal gels were employed as a separations matrix because of their unique ability to change viscosity in response to temperature. Proteins were added into thermal gel and loaded into a microfluidic device. Electrolyte optimization was conducted to achieve robust tITP to isotachophoretically preconcentrate proteins and then electrophoretically separate them. Electropherograms were collected through both time and distance to enable both small and large proteins to be measured within a single analysis. The effects of temperature were evaluated and found to exhibit a pronounced effect on the separation. Temperature gradients were then employed to alter thermal gel viscosity over time to maximize separation resolution between proteins. The results herein demonstrate how gradient TG-tITP achieves rapid, high-performance separations of native proteins. This analysis provided a wide mass range (6–464 kDa) with two-fold higher resolution than native PAGE while requiring 15,000-fold less protein loading and providing five-fold faster analysis times. Graphical abstract

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: Analysis / Analytical Chemistry / Biochemistry / Characterization and Evaluation of Materials / Chemistry / Chemistry and Materials Science / electrolytes / Electrophoresis / Electrophoresis, Polyacrylamide Gel / Food Science / Gels / Isotachophoresis / Isotachophoresis - methods / Laboratories / Laboratory Medicine / Methods / Microfluidic devices / Microfluidics / Monitoring/Environmental Analysis / Native Polyacrylamide Gel Electrophoresis / Optimization / Paper in Forefront / Polyacrylamide / polyacrylamide gel electrophoresis / Protein folding / Proteins / Proteins - chemistry / Separation / Separation (Technology) / Temperature / Temperature effects / Temperature gradients / Viscosity / Young Investigators in (Bio-)Analytical Chemistry 2023