Separating Buffer, also known as Resolving Buffer, is primarily used in polyacrylamide gel electrophoresis (PAGE) for the separation of proteins based on their molecular weight. It creates a pH environment that ensures proteins remain negatively charged, allowing them to migrate through the gel matrix toward the anode. This buffer is critical in the resolving gel, where proteins are separated into distinct bands according to their size. It is widely applied in molecular biology and biochemistry research for analyzing protein mixtures, assessing purity, and studying protein-protein interactions. Additionally, it plays a key role in techniques like SDS-PAGE, enabling accurate determination of protein molecular weights.