Nanoparticles (NPs) are able to improve the separation efficiency of proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) due to their capability of enhancing heat dissipation during electrophoresis. However, the intrinsic surface charges of NPs (at buffer pH or charge induced due to the SDS coating) make them acquire electrophoretic mobility and movement in the gel. Such a movement leads to viscosity and temperature gradients in the gel and deteriorates the separation. In this work, we proposed a novel method by using tethered NPs in the gel. Silica NPs, as the model NPs, were prepared and their surfaces were modified using 3-[(methacryloxy)propyl] trimethoxysilane (MPS) which locks the NPs in the gel via covalent bonds (M-SiO2/PA (polyacrylamide)). SiO2 NPs were embedded into the gel (SiO2/PA) as the positive control, while pure PA gel was chosen as the negative control. The results showed that at a relatively high voltage of 250 V, although the Joule heat generated during electrophoresis disturbed the separation in the pure gel, the SiO2/PA and M-SiO2/PA nanocomposite gels showed better performances. In comparison with the pure PA gel, the resolution increased by 3 and 32% for SiO2/PA and M-SiO2/PA, respectively, in a relatively short separation time of 35 min. The gel with tethered NPs presented a more efficient separation in terms of band broadening and resolution compared with the gel with free NPs probably due to the movement of free charged particles in the gel. Evidently, the migration speed of protein bands in the gels decreased especially for larger proteins in the presence of the NPs compared to the pristine gel due to the steric hindrance of the NPs. Finally, we separated E. coli proteins, as a real sample. Among the three gels (pure PA, SiO2/PA, and M-SiO2/PA), the gel containing M-SiO2 showed the best performance.

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