Why Does the Protein Band Broaden After Moving from Stacking to Separating Gel in SDS-PAGE
In the process of SDS-PAGE electrophoresis, protein samples are first loaded onto the stacking gel and then migrate into the separating gel. These two types of gels serve different functions and possess distinct physicochemical properties:
1. Stacking Gel
This gel is characterized by a low polyacrylamide concentration and relatively large pore size. It does not separate proteins based on molecular weight. Instead, its primary function is to concentrate all proteins into a sharply focused zone, thereby forming a well-defined starting point. This concentration effect enhances the resolution of protein separation in the subsequent separating gel.
2. Separating Gel
This gel has a higher polyacrylamide concentration and smaller pore size. As proteins migrate from the stacking gel into the separating gel, they begin to separate according to their molecular weights. Smaller proteins travel faster through the gel matrix, whereas larger proteins migrate more slowly. As a result, the initially compressed protein sample, which appeared as a narrow band in the stacking gel, becomes spatially resolved into distinct protein bands in the separating gel.
Therefore, after being concentrated into a single zone within the stacking gel, the proteins undergo size-based separation in the separating gel, leading to the appearance of distinct bands corresponding to different molecular weights on the electrophoretic gel.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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