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When the proteins in sample buffer are loaded onto the gel and an electric current is applied, they get trapped in what is termed the moving boundary while migrating through the stacker. Chloride ions from the Tris-HCl in the stacker and the sample buffer form the front part of this boundary, while glycine molecules from the running bufferform the back part of this boundary. The proteins get sandwiched between the chloride ions and the glycine molecules and form a very thin zone, or stack. When this moving boundary reaches the resolving portion of the gel, the difference in pH
between the stacker and the separator causes the glycine molecules to ionize and the glycine ions move through the protein stack right behind the chloride ions. Freed from the moving boundary, theproteins move through the separator, the distance covered being dictated by the size of the protein and the size of the pores in the separator.
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Separating gel allows the separation of protein molecules according to their molecular weight by sieving effect of pores in the gel(percentage). The pH of separating or resolving gel is 8.8, whereas stacking gel (upper gel that squeezes protein as a thin layer) made of pH6.8.
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