What is the pH of resolving gel in SDS-PAGE?
SDS-PAGE employs a discontinuous buffer system with distinct pH zones to achieve stacking and separation. Stacking gel is cast at pH 6.8 in Tris-HCl to keep glycine largely as zwitterion with low electrophoretic mobility, enabling isotachophoretic concentration of proteins between chloride leading ions and glycine trailing ions. Resolving gel is cast at pH 8.8, where glycine acquires full negative charge, moves rapidly ahead, and allows proteins to separate by molecular sieving through smaller pores. This alkaline pH maintains consistent Tris buffering and stable migration. Values like 6.8, 7.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.