Role of Glycine in Protein Separation in SDS‐Polyacrylamide Gel Electrophoresis: Gel Synthesis, Characterizations, and Performance

ABSTRACT Electrophoresis, a fundamental technique for separating charged biomolecules such as proteins and DNA, operates on the principles involving molecular weight, electrical charge, and the properties of the separation medium. Polyacrylamide gel electrophoresis (PAGE) is a versatile tool widely utilized to separate and analyze proteins. This study investigates the role of glycine in polyacrylamide gels to potentially enhance protein separation performance. Two distinct methods of incorporating glycine into polyacrylamide gel are explored: Pre‐electrophoresis and direct mixing . Scanning electron microscopy and rheological tests are used to characterize the internal structure and mechanical properties of the gels, respectively. The performance of the modified polyacrylamide–glycine gel and the enhancement of its molecular sieving capacity are investigated by testing the gel's ability to separate proteins in a protein ladder with varying glycine levels and/or polymer percentages. Incorporation of glycine by either method yields comparable enhancements in protein separation. Direct mixing of glycine with gel solutions requires specific concentrations (192 m M for 6% gels and 400 m M for 4% gels) to achieve the performance of pre‐electrophoresis‐treated gels. Glycine improves protein separation, with corresponding mechanical and structural changes revealed in the modified gels. The methods investigated offer a potential new approach for improving PAGE‐based separation of protein mixtures.