Separation processes are routinely used worldwide in biotechnology, chemical processing, wastewater treatment, and myriad other areas. Gel electrophoresis has been used for decades as a standard technique to separate charged biopolymers, such as DNA, RNA, and proteins. In this research, polyacrylamide hydrogels were synthesized in the presence of sodium dodecyl sulfate (SDS) micelles as nanotemplating agents in an effort to modify the internal porous microstructure of the gels. After removing these agents via a combination of passive and facilitated means, the gels were used during electrophoresis in order to assess the effects of its modified porous microstructure on protein separations. The results revealed that hydrogels containing 9% acrylamide and templated with SDS micelles in a 5–15% concentration range were the most effective materials in separating proteins in a range of 10–250 kDa. As expected, standard, nontemplated gels also resulted in separation of the proteins but not to the same extent as with the templated hydrogels. In summary, this research highlights the important role of the templating agent as possible useful tuning factors for achieving electrophoresis-based separation. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 44063.