Rheological and mechanical behavior of polyacrylamide hydrogels chemically crosslinked with allyl agarose for two-dimensional gel electrophoresis

Two-dimensional (2-D) gel electrophoresis currently represents one of the most standard techniques for protein separation. In addition to the most commonly employed polyacrylamide crosslinked hydrogels, acrylamide–agarose copolymers have been proposed as promising systems for separation matrices in 2-D electrophoresis, because of the good resolution of both high and low molecular mass proteins made possible by careful control and optimization of the hydrogel pore structure. As a matter of fact, a thorough understanding of the nature of the hydrogel pore structure as well as of the parameters by which it is influenced is crucial for the design of hydrogel systems with optimal sieving properties. In this work, a series of acrylamide-based hydrogels covalently crosslinked with different concentrations of allyl agarose (0.2–1%) is prepared and characterized by creep-recovery measurements, dynamic rheology and tensile tests, in the attempt to gain a clearer understanding of structure–property relationships in crosslinked polyacrylamidebased hydrogels. The rheological and mechanical properties of crosslinked acrylamide– agarose hydrogels are found to be greatly affected by crosslinker concentration. Dynamic rheological tests show that hydrogels with a percentage of allyl agarose between 0.2% and 0.6% have a low density of elastically effective crosslinks, explaining the good separation of high molecular mass proteins in 2-D gel electrophoresis. Over the same range of crosslinker concentration, creep-recovery measurements reveal the presence of non-permanent crosslinks in the hydrogel network that justifies the good resolution of low molecular mass proteins as well. In tensile tests, the hydrogel crosslinked with 0.4% of allyl agarose exhibits the best results in terms of mechanical strength and toughness. Our results show how the control of the viscoelastic and the mechanical properties of these materials allow the design of mechanically stable hydrogels with improved sieving ability in protein electrophoresis over a wide range of molecular masses.