Hello Sangramjit Basak

The difference in pH between the stacking and resolving gels is to concentrate the protein sample into a narrow band at the interface between the gels before they are separated in the resolving gel.

The difference in pH between the stacking and resolving gels allows glycine, which is used in the running buffer, to exhibit distinct properties. In the stacking gel (pH 6.8), glycine is primarily in its zwitterionic form, moving slowly. Simultaneously, chloride ions from the Tris-HCl buffer move much faster. The proteins, with their intermediate mobility, get sandwiched between the slowly moving glycine and the rapidly moving chloride ions, forming a thin band in the stacking gel. This phenomenon is called stacking, and it ensures that all proteins enter the resolving gel simultaneously.

As the glycine-protein band enters the higher pH (pH 8.8) in the resolving gel, this pH shift causes glycine to become negatively charged (glycinate). This anionic form of glycine moves rapidly in the electric field, effectively leading the way. Since the proteins are also negatively charged due to SDS, they move in the electric field based on their molecular size, allowing them to separate according to their molecular weight.

So, having two different pH in SDS-PAGE for stacking and resolving gels is essential because it will allow glycine to first concentrate the proteins, and then with the higher pH in the resolving gel, it will allow glycine to move rapidly past the proteins enabling the separation of proteins based on size in the resolving gel.

Best,

In SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis), the use of two different pH values for the stacking and resolving gels is a fundamental feature of the discontinuous buffer system, originally described by Laemmli (1970). The stacking gel typically operates at a pH of approximately 6.8, while the resolving (or separating) gel functions at a higher pH of around 8.8. This pH differentiation plays a critical role in enhancing the resolution and sharpness of protein bands. In the stacking gel, the lower pH maintains glycine (from the running buffer) in a predominantly zwitterionic state, which migrates more slowly than the chloride ions present in the gel. Consequently, proteins—coated with SDS to ensure a uniform negative charge-to-mass ratio—become sandwiched between the fast-moving chloride front and the slower-moving glycine ions. This ion arrangement generates a voltage discontinuity that compresses proteins into a thin, concentrated zone, a process termed "stacking" (Laemmli, 1970; Walker, 2009).

Upon entering the resolving gel at pH 8.8, glycine becomes fully deprotonated and negatively charged, accelerating its migration. This removes the stacking effect and allows proteins to migrate according to size through the polyacrylamide matrix, where smaller proteins traverse the gel faster than larger ones. The higher pH also enhances the buffering capacity and sharpens band resolution. The gel composition, including acrylamide concentration and buffer pH, thus plays a pivotal role in modulating electrophoretic behavior and achieving precise molecular weight-based separation of proteins (Laemmli, 1970; Kurien & Scofield, 2012).

References:

· Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. https://doi.org/10.1038/227680a0

· Walker, J. M. (2009). The Protein Protocols Handbook (3rd ed.). Humana Press. https://doi.org/10.1007/978-1-59745-198-7

· Kurien, B. T., & Scofield, R. H. (2012). Protein blotting: A review. Journal of Immunological Methods, 274(1–2), 1–15. https://doi.org/10.1016/S0022-1759(02)00378-7