Question

Biochem Lab: Explain the basis for the stacking gel portion of a polyacrylamide gel. Describe how it works with respect to the loaded protein samples as well as the running buffer components.

Answer 1

Polyacrylamide gel is commonly used in the lab for the separation of proteins based on their molecular weight. It’s one of the most commonly technique used but a tedious process.

It separates proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field. The phenomenon is known as electrophoresis.

The natively folded proteins neither have net charge nor their molecular radius is molecular weight dependent. So this is problem associated with the tertiary structure of the proteins unlike any charged species whose movement can be monitored by the quantity of their net charge, molecular radius and magnitude of applied field. So in case of proteins, Instead, their net charge is determined by amino acid composition i.e. the sum of the positive and negative amino acids in the protein and molecular radius by the protein’s tertiary structure.So in their native state, different proteins with the same molecular weight would migrate at different speeds in an electrical field depending on their charge and 3D shape.

In conclusion to separate proteins in an electrical field based on their molecular weight only, we need to destroy the tertiary structure by reducing the protein to a linear molecule, and somehow mask the intrinsic net charge of the protein. That’s where polyacrylamide gel comes in.

Polyacrylamide with a bit of boiling, disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules and their charges masked, they stay that way throughout the run. Also it binds uniformly to the linear proteins meaning that the charge of the protein is now approximately equal to its molecular weight.

In an applied electrical field, the polyacrylamide-treated proteins will now move toward the positive anode at different rates depending on their molecular weight. These different mobilities will be exaggerated due to the high-friction environment of a gel matrix.

As the name suggests, the gel matrix is polyacrylamide, which is a good choice because it is chemically inert and, crucially, can easily be made up at a variety concentrations to produce different pore sizes giving a variety of separating conditions that can be changed depending on your needs.

Various buffer systems are used in PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different.

Typically, the system is set up with a stacking gel at pH 6.8, buffered by Tris-HCl, a running gel buffered to pH 8.8 by Tris-HCl and an electrode buffer at pH 8.3. The stacking gel has a low concentration of acrylamide and the running gel a higher concentration capable of retarding the movement of the proteins.

Glycine can exist in three different charge states, positive, neutral or negative, depending on the pH. This is shown in the diagram below. Control of the charge state of the glycine by the different buffers is the key to the whole stacking gel thing.

When the power is turned on, the negatively-charged glycine ions in the pH 8.3 electrode buffer are forced to enter the stacking gel, where the pH is 6.8. In this environment, glycine switches predominantly to the zwitterionic (neutrally charged) state. This loss of charge causes them to move very slowly in the electric field.

The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in the electric field and they form an ion front that migrates ahead of the glycine. The separation of Cl- from the Tris counter-ion (which is now moving towards the anode) creates a narrow zone with a steep voltage gradient that pulls the glycine along behind it, resulting in two narrowly separated fronts of migrating ions; the highly mobile Cl- front, followed by the slower, mostly neutral glycine front.

All of the proteins in the gel sample have an electrophoretic mobility that is intermediate between the extreme of the mobility of the glycine and Cl-, so when the two fronts sweep through the sample well, the proteins are concentrated into the narrow zone between the Cl- and glycine fronts.